Nucleic acid encoding a retinoblastoma binding protein (RBP-7) and polymorphic markers associated with said nucleic acid

ABSTRACT

The present invention is directed to a polynucleotide comprising open reading frames defining a coding region encoding a retinoblastoma binding protein (RBP-7) as well as regulatory regions located both at the 5′ end and the 3′ end of said coding region. The present invention also pertains to a polynucleotide carrying the natural regulation signals of the RBP-7 gene which is useful in order to express a heterologous nucleic acid in host cells or host organisms as well as functionally active regulatory polynucleotides derived from said regulatory region. The invention also concerns polypeptides encoded by the coding region of the RBP-7 gene. The invention also deals with antibodies directed specifically against such polypeptides that are useful as diagnostic reagents. The invention also comprises genetic markers, namely biallelic markers, that are means that may be useful for the diagnosis of diseases related to an alteration in the regulation or in the coding regions of the RBP-7 gene and for the prognosis/diagnosis of an eventual treatment with therapeutic agents, especially agents acting on pathologies involving abnormal cell proliferation and/or abnormal cell differentiation.

RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application Serial No. 60/091,315, filed Jun. 30, 1998 and U.S. Provisional Patent Application Serial No. 60/111,909, filed Dec. 10, 1998, the disclosures of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

[0002] The present invention is directed to a polynucleotide comprising open reading frames defining a coding region encoding a retinoblastoma binding protein (RBP-7) as well as regulatory regions located both at the 5′ end and the 3′ end of said coding region. The present invention also pertains to a polynucleotide carrying the natural regulation signals of the RBP-7 gene which is useful in order to express a heterologous nucleic acid in host cells or host organisms as well as functionally active regulatory polynucleotides derived from said regulatory region. The invention also concerns polypeptides encoded by the coding region of the RBP-7 gene. The invention also deals with antibodies directed specifically against such polypeptides that are useful as diagnostic reagents. The invention includes genetic markers, namely biallelic markers, that are means that may be useful for the diagnosis of diseases related to an alteration in the regulation or in the coding regions of the RBP-7 gene and for the prognosis/diagnosis of an eventual treatment with therapeutic agents, especially agents acting on pathologies involving abnormal cell proliferation and/or abnormal cell differentiation.

BACKGROUND OF THE INVENTION

[0003] Among the genetic alterations that have been shown to represent direct or indirect causative agents of proliferative diseases, such as cancers, there may be cited mutations occurring at loci harboring genes that are called tumor suppressor genes.

[0004] Tumor suppressor genes are defined as genes involved in the control of abnormal cell proliferation and whose loss or inactivation is associated with the development of malignancy. Tumor suppressor genes encompass ortho-genes, emerogenes, flatogenes, and onco-supressor genes.

[0005] More specifically, tumor suppressor genes are genes whose products inhibit cell growth. Mutant alleles in cancer cells have lost their normal function, and act in the cell in a recessive way in that both copies of the gene must be inactivated in order to change the cell phenotype. The tumor phenotype can be rescued by the wild-type allele, as shown by cell fusion experiments first described by Harris and colleagues (Harris H. et al., 1969). Germline mutations of tumor suppressor genes may be transmitted and thus studied in both constitutional and tumor DNA from familial or sporadic cases. The current family of tumor suppressors include DNA-binding transcription factors (i.e. p53, WT1), transcription regulators (i.e., RB, APC) and protein kinase inhibitors (i.e. p16).

[0006] The existence of tumor suppressor genes has been particularly shown in cases of hereditary cancers. These are cancer where there is a clear pattern of inheritance, usually autosomal dominant, with a tendency for earlier age of onset than for sporadic tumors.

[0007] Tumor suppressor genes are detected in the form of inactivating mutations that are tumorigenic. The two best characterized genes of this class code for the proteins RB (Retinoblastoma protein) and p53.

[0008] Retinoblastoma is a human childhood disease, involving a tumor in the retina. It occurs both as an inheritable trait and sporadically (by somatic mutation). Retinoblastoma arises when both copies of the RB gene are inactivated. In the inherited form of the disease, one parental chromosome carries an alteration in this region, usually a deletion. A somatic event in retinal cells that causes the loss of the other copy of the RB gene causes a tumor. Forty percent of cases are hereditary, transmitted as an autosomal dominant trait with 90% penetrance. Of these cases, around 10-15% are transmitted from an affected parent, the remaining arising as de novo germ-lime mutations. In the sporadic form of the disease, the parental chromosomes are normal, and both RB alleles are lost by somatic events. The tumor suppressor nature of RB was shown by the introduction of a single copy of RB1 into tumor cell lines lacking the gene, resulting in complete or partial suppression of the tumorigenic phenotype.

[0009] The RB protein has a regulatory role in cell proliferation, acting via transcription factors to prevent the transcriptional activation of a variety of genes, the products of which are required for the onset of DNA synthesis, the S phase of the cell cycle.

[0010] When investigating on the molecular function of RB, it has been found that the RB protein interacts with a variety of viral proteins, including several tumor antigens, such as SV40 T antigen, adenovirus E1A protein, human papillomavirus E7. These viral proteins have been shown to bind to RB, thereby inactivating it and allowing cell division to occur.

[0011] Thus, an important step toward defining a mechanism underlying tumor suppressor activity of the RB gene was the observation that the transforming products of adenovirus (E1A protein), simian virus 40 (large T antigen) and human papillomavirus (E7 protein) could precipitate wild-type RB protein. This, in turn, led to the identification of a family of cellular proteins that can reversibly bind to a discrete domain on the RB protein, referred to as the T/E1A pocket by using the same specificity as the viral products. The subsequent observation that protein binding was inhibited following RB protein phosphorylation in the late G₁ phase of the cell cycle suggested the hypothesis that the RB protein, as well as the related product p107, may regulate the functional activity of its binding partners by a cell-cycle dependent pattern of physical association. In particular, the activity of the RB protein has been shown to be regulated through cell cycle-dependent phosphorylation by cyclin-dependent kinases.

[0012] The picture of transcription regulation is made even more complex by the finding that a number of RB related proteins (e.g. p107 and p130) also bind members of the E2F family and are therefore involved in regulatory process.

[0013] In view of the foregoing, there clearly exists a pressing need to identify and characterize the cellular proteins that interact with the retinoblastoma protein in order to provide diagnostic and therapeutic tools useful to prevent and cure cell differentiation disorders, particularly disorders in which a lack of completion of cell differentiation , particularly in terminal cell differentiation, or in which an abnormal cell proliferation is detected, such as in proliferative diseases like cancer.

[0014] For the purpose of the present invention, cells with abnormal proliferation include, but are not limited to, cells characteristic of the following disease states: thyroid hyperplasia, psoriasis, benign prostatic hypertrophy, cancers including breast cancer, sarcomas and other neoplasms, bladder cancer, colon cancer, lung cancer, prostate cancer, various leukemias and lymphomas.

SUMMARY OF THE INVENTION

[0015] This invention is based on the discovery of a nucleic acid molecule encoding a novel protein, more particularly a retinoblastoma binding protein (RBP-7).

[0016] The present invention pertains to nucleic acid molecules comprising the genomic sequence of the gene encoding RBP-7. The RBP-7 genomic sequence comprises regulatory sequence located upstream (5′-end) and downstream (3′-end) of the transcribed portion of said gene, these regulatory sequences being also part of the invention.

[0017] The invention also deals with the complete cDNA sequence encoding the RBP-7 protein, as well as with the corresponding translation product.

[0018] Oligonucleotide probes or primers hybridizing specifically with a RBP-7 genomic or cDNA sequence are also part of the present invention, as well as DNA amplification and detection methods using said primers and probes.

[0019] A further aspect of the invention is recombinant vectors comprising any of the nucleic acid sequences described above, and in particular of recombinant vectors comprising a RBP-7 regulatory sequence or a sequence encoding a RBP-7 protein, as well as of cell hosts and transgenic non human animals comprising said nucleic acid sequences or recombinant vectors.

[0020] Finally, the invention is directed to methods for the screening of substances or molecules that inhibit the expression of RBP-7, as well as with methods for the screening of substances or molecules that interact with a RBP-7 polypeptide or that modulate the activity of a RBP-7 polypeptide.

[0021] The invention also concerns biallelic markers of the RBP-7 gene which can be useful for genetic studies, for diagnosis of diseases related to an alteration in the regulation or in the coding regions of the RBP-7 gene and for the prognosis/diagnosis of an eventual treatment with therapeutic agents, especially agents acting on pathologies involving abnormal cell proliferation and/or abnormal cell differentiation

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]FIG. 1 is a diagram showing a map of the RBP-7 gene.

[0023]FIG. 2 is a presentation of the RBP-7 gene structure with the amplified fragments and the biallelic markers of the present invention.

BRIEF DESCRIPTION OF THE SEQUENCES PROVIDED IN THE SEQUENCE LISTING

[0024] SEQ ID No. 1 contains a genomic sequence of RBP-7 comprising the 5′ regulatory region (upstream untranscribed region), the exons and introns, and the 3′ regulatory region (downstream untranscribed region).

[0025] SEQ ID No. 2 contains the 5′-regulatory sequence (upstream untrancribed region) of RBP-7.

[0026] SEQ ID No. 3 contains the 3′-regulatory sequence (upstream untrancribed region) of RBP-7.

[0027] SEQ ID No. 4 contains the RBP-7 cDNA sequence.

[0028] SEQ ID Nos 5 to 28 contain the exons 1 to 24 of RBP-7.

[0029] SEQ ID No. 29 contains the protein sequence encoded by the nucleotide sequence of SEQ ID No. 4.

[0030] SEQ ID Nos 30 to 50 contain the fragments containing a polymorphic base of a biallelic marker (first allele).

[0031] SEQ ID Nos 51 to 71 contain the fragments containing a polymorphic base of a biallelic marker (second allele).

[0032] SEQ ID Nos 72 to 101 contain the amplification primers.

[0033] SEQ ID Nos 102 to 136 contain the microsequencing primers.

[0034] SEQ ID Nos 137 and 138 contain cDNA amplification primers.

[0035] SEQ ID Nos 139 and 140 respectively contain a primer containing the additional PU 5′ sequence and the additional RP 5′ sequence described further in Example 3.

[0036] In accordance with the regulations relating to Sequence Listings, the following codes have been used in the Sequence Listing to indicate the locations of biallelic markers within the sequences and to identify each of the alleles present at the polymorphic base. The code “r” in the sequences indicates that one allele of the polymorphic base is a guanine, while the other allele is an adenine. The code “y” in the sequences indicates that one allele of the polymorphic base is a thymine, while the other allele is a cytosine. The code “m” in the sequences indicates that one allele of the polymorphic base is an adenine, while the other allele is an cytosine. The code “k” in the sequences indicates that one allele of the polymorphic base is a guanine, while the other allele is a thymine. The code “s” in the sequences indicates that one allele of the polymorphic base is a guanine, while the other allele is a cytosine. The code “w” in the sequences indicates that one allele of the polymorphic base is an adenine, while the other allele is an thymine. The nucleotide code of the original allele for each biallelic marker is the following: Biallelic marker Original allele  5-124-273 A  5-127-261 C  5-130-257 A  5-130-276 A  5-131-395 A  5-135-357 A  5-136-174 T  5-140-120 T  5-143-101 C  5-143-84 G  5-145-24 A  5-148-352 T 99-1437-325 A 99-1442-224 T

[0037] In some instances, the polymorphic bases of the biallelic markers alter the identity of an amino acids in the encoded polypeptide. This is indicated in the accompanying Sequence Listing by use of the feature VARIANT, placement of an Xaa at the position of the polymorphic amino acid, and definition of Xaa as the two alternative amino acids. For example if one allele of a biallelic marker is the codon CAC, which encodes histidine, while the other allele of the biallelic marker is CAA, which encodes glutamine, the Sequence Listing for the encoded polypeptide will contain an Xaa at the location of the polymorphic amino acid. In this instance, Xaa would be defined as being histidine or glutamine.

[0038] In other instances, Xaa may indicate an amino acid whose identity is unknown. In this instance, the feature UNSURE is used, placement of an Xaa at the position of the unknown amino acid and definition of Xaa as being any of the 20 amino acids or being unknown.

DETAILED DESCRIPTION OF THE INVENTION

[0039] The aim of the present invention is to provide polynucleotides and polypeptides related to the RBP-7 gene and to a RBP-7 protein, which is potentially involved in the regulation of the differentiation of various cell types in mammals. A deregulation or an alteration of this protein may be involved in the generation of a pathological state in a patient. Such pathological state includes disorders caused by cell apoptosis or in contrast by an abnormal cell proliferation such as in cancers.

[0040] The unphosphorylated form of the Retinoblastoma (RB) protein specifically binds several proteins, and these interactions occur only during part of the cell cycle, prior to the S phase. The target proteins of the RB protein include E2F transcription factors and cyclins of the D and E types. Binding to the RB protein inhibits the ability of E2F to activate transcription, which suggests that the RB protein may repress the expression of genes dependent on E2F. Interaction of the RB protein with E2F-1, a member of the E2F transcription factors family, inhibits transcription of genes involved in DNA synthesis and therefore suppresses cell growth. Additionally, it has been found that the complexes formed between E2F and the RB protein are disrupted in the presence of the viral oncoproteins that bind to the RB protein, suggesting a key role of the RB protein in the regulation of E2F activity.

[0041] It has been shown that the RB protein forms two types of complexes with E2F. One of these two types involves a binary complex of the RB protein and E2F that does not bind DNA in a gel retardation assay, and the second type of RB protein/E2F complex involves another factor, RBP60, which allows the RB protein/E2F complex to bind DNA and produce a distinct complex in a gel retardation assay. One hypothesis is that RB protein might be regulating the DNA-binding as well as the transcription activation function of E2F. It has also been demonstrated that E2F can bind DNA as an oligomeric complex composed of at least two distinct proteins.

[0042] Recent reports indicate that approximately 10 proteins have been identified that bind to the RB protein using the same binding surface as the viral oncoproteins. Several of these cellular proteins, including the E2F transcription factor described above, comprise members of the myc oncogene family, a p46 protein (Rb-AP46), MyoD, Elf-1, protein phosphatase type 1 catalytic subunit and several proteins designated generically as “Retinoblastoma Binding Proteins” (RBBP), some of these latter proteins being defined as E2F-like proteins.

[0043] Defeo-Jones et al. (1991) have cloned the cDNA of two members of the RBBP family, namely RBP-1 and RBP-2. RBP-1 and RBP-2 bind specifically to the RB protein in vitro. RBP-2 has been shown to interact noncovalently with RB ptotein via the binding of a consensus amino acid sequence of RBP-2, namely the LXCXE amino acid sequence, to the conserved T/EIA pocket of the RB protein (Kim et al., 1994). This LXCXE consensus amino acid sequence is also present within the adenovirus E1A protein, the SV40 large T antigen as well as within the human papillomavirus E7 protein. RBP-1 and RBP-2 have been hypothesized to function as transcription factors, like E2F. Helin et al. (1992) have cloned a cDNA encoding another member of the RBBP family, namely RBP-3. Sakai et al. (1995) have cloned a novel RBBP protein designated as RBP-6, the locus of which has been mapped on chromosome 16 between p11.2 and p12.

[0044] For the E2F family, replicating and differentiating cells need the RB protein or RB protein family members (e.g. p107 or p130) to counterbalance its apoptotic effect. E2F induces apoptosis when over-expressed in cells with the wild type p53 gene, but favors proliferation in p53-/- cells. E2F-induced apoptosis follows entry of the cell into S-phase. The E2F death-promoting effect can be blocked by co-expression of p105, a RB protein family member. Conversely, by gene knock-out studies, it has been demonstrated that E2F is critical for the normal development of diverse cell types. Mice null for the E2F1 gene show defects at a young age in the terminal differentiation of cell types in which apoptosis play an important role, namely T-cells or epithelial cells of the testis or of other exocrine glands. With increasing age, these animals develop wide-spread tumors. This data indicates that E2F plays a physiological role in normal development, probably by inducing apoptosis in a specific set of developing cells.

[0045] The retinoblastoma binding proteins of the E2F type have also been described in PCT Application No. WO 65/24223, PCT Application No. WO 96/25494 and in U.S. Pat. No. 5,650,287, the disclosures of which are incorporated herein by reference in their entireties. Other retinoblastoma binding proteins have been described, notably in PCT Application No. WO 94/12521 , in PCT Application No. WO 95/17198, in PCT Application No. 93/23539 and in PCT Application No. WO 93/06168, the disclosures of which are incorporated herein by reference in their entireties.

Definitions

[0046] Before describing the invention in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used to describe the invention herein.

[0047] The term “RBP-7 gene”, when used herein, encompasses mRNA and cDNA sequences encoding the RBP-7 protein. In the case of a genomic sequence, the RBP-7 gene also includes native regulatory regions which control the expression of the coding sequence of the RBP-7 gene.

[0048] The term “functionally active fragment” of the RBP-7 protein is intended to designate a polypeptide carrying at least one of the structural features of the RBP-7 protein involved in at least one of the biological functions and/or activity of the RBP-7 protein. Particularly preferred are peptide fragments carrying either the retinoblastoma protein binding domain and/or the DNA binding domain of the RBP-7 protein.

[0049] A “heterologous” or “exogenous” polynucleotide designates a purified or isolated nucleic acid that has been placed, by genetic engineering techniques, in the environment of unrelated nucleotide sequences, such as the final polynucleotide construct does not occur naturally. An illustrative, but not limitatitive, embodiment of such a polynucleotide construct may be represented by a polynucleotide comprising (1) a regulatory polynucleotide derived from the RBP-7 gene sequence and (2) a polynucleotide encoding a cytokine, for example GM-CSF. The polypeptide encoded by the heterologous polynucleotide will be termed an heterologous polypeptide for the purpose of the present invention.

[0050] By a “biologically active fragment or variant” of a regulatory polynucleotide according to the present invention is intended a polynucleotide comprising or alternatively consisting of a fragment of said polynucleotide which is functional as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide in a recombinant cell host.

[0051] For the purpose of the invention, a nucleic acid or polynucleotide is “functional” as a regulatory region for expressing a recombinant polypeptide or a recombinant polynucleotide if said regulatory polynucleotide contains nucleotide sequences which contain transcriptional and translational regulatory information, and such sequences are “operatively linked” to nucleotide sequences which encode the desired polypeptide or the desired polynucleotide. An operable linkage is a linkage in which the regulatory nucleic acid and the DNA sequence sought to be expressed are linked in such a way as to permit gene expression.

[0052] As used herein, the term “operably linked” refers to a linkage of polynucleotide elements in a functional relationship. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence. More precisely, two DNA molecules (such as a polynucleotide containing a promoter region and a polynucleotide encoding a desired polypeptide or polynucleotide) are said to be “operably linked” if the nature of the linkage between the two polynucleotides does not (1) result in the introduction of a frame-shift mutation or (2) interfere with the ability of the polynucleotide containing the promoter to direct the transcription of the coding polynucleotide. The promoter polynucleotide would be operably linked to a polynucleotide encoding a desired polypeptide or a desired polynucleotide if the promoter is capable of effecting transcription of the polynucleotide of interest.

[0053] An “altered copy” of the RBP-7 gene is intended to designate a RBP-7 gene that has undergone at least one substitution, addition or deletion of one or several nucleotides, wherein said nucleotide substitution, addition or deletion preferably causes a change in the amino acid sequence of the resulting translation product or alternatively causes an increase or a decrease in the expression of the RPB-7 gene.

[0054] The terms “sample” or “material sample” are used herein to designate a solid or a liquid material suspected to contain a polynucleotide or a polypeptide of the invention. A solid material may be, for example, a tissue slice or biopsy which is searched for the presence of a polynucleotide encoding a RBP-7 protein, either a DNA or RNA molecule or within which is searched for the presence of a native or a mutated RBP-7 protein, or alternatively the presence of a desired protein of interest the expression of which has been placed under the control of a RBP-7 regulatory polynucleotide. A liquid material may be, for example, any body fluid like serum, urine etc., or a liquid solution resulting from the extraction of nucleic acid or protein material of interest from a cell suspension or from cells in a tissue slice or biopsy. The term “biological sample” is also used and is more precisely defined within the Section dealing with DNA extraction.

[0055] As used herein, the term “purified” does not require absolute purity; rather, it is intended as a relative definition. Purification if starting material or natural material to at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated. As an example, purification from 0.1% concentration to 10% concentration is two orders of magnitude.

[0056] The term “isolated” requires that the material be removed from its original environment (e.g. the natural environment if it is naturally occurring). For example, a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or DNA or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotide could be part of a vector and/or such polynucleotide or polypeptide could be part of a composition and still be isolated in that the vector or composition is not part of its natural environment.

[0057] Throughout the present specification, the expression “nucleotide sequence” may be employed to designate indifferently a polynucleotide or an oligonucleotide or a nucleic acid. More precisely, the expression “nucleotide sequence” encompasses the nucleic material itself and is thus not restricted to the sequence information (i.e. the succession of letters chosen among the four base letters) that biochemically characterizes a specific DNA or RNA molecule.

[0058] As used interchangeably herein, the term “oligonucleotides”, and “polynucleotides” include RNA, DNA, or RNA/DNA hybrid sequences of more than one nucleotide in either single chain or duplex form. The term “nucleotide” as used herein as an adjective to describe molecules comprising RNA, DNA, or RNA/DNA hybrid sequences of any length in single-stranded or duplex form. The term “nucleotide” is also used herein as a noun to refer to individual nucleotides or varieties of nucleotides, meaning a molecule, or individual unit in a larger nucleic acid molecule, comprising a purine or pyrimidine, a ribose or deoxyribose sugar moiety, and a phosphate group, or phosphodiester linkage in the case of nucleotides within an oligonucleotide or polynucleotide. Although the term “nucleotide” is also used herein to encompass “modified nucleotides” which comprise at least one modifications (a) an alternative linking group, (b) an analogous form of purine, (c) an analogous form of pyrimidine, or (d) an analogous sugar, for examples of analogous linking groups, purine, pyrimidines, and sugars see for example PCT publication No. WO 95/04064. However, the polynucleotides of the invention are preferably comprised of greater than 50% conventional deoxyribose nucleotides, and most preferably greater than 90% conventional deoxyribose nucleotides. The polynucleotide sequences of the invention may be prepared by any known method, including synthetic, recombinant, ex vivo generation, or a combination thereof, as well as utilizing any purification methods known in the art.

[0059] The term “heterozygosity rate” is used herein to refer to the incidence of individuals in a population which are heterozygous at a particular allele. In a biallelic system, the heterozygosity rate is on average equal to 2P_(a)(1−P_(a)), where P_(a) is the frequency of the least common allele. In order to be useful in genetic studies, a genetic marker should have an adequate level of heterozygosity to allow a reasonable probability that a randomly selected person will be heterozygous.

[0060] The term “genotype” as used herein refers the identity of the alleles present in an individual or a sample. In the context of the present invention a genotype preferably refers to the description of the biallelic marker alleles present in an individual or a sample. The term “genotyping” a sample or an individual for a biallelic marker consists of determining the specific allele or the specific nucleotide carried by an individual at a biallelic marker.

[0061] The term “polymorphism” as used herein refers to the occurrence of two or more alternative genomic sequences or alleles between or among different genomes or individuals. “Polymorphic” refers to the condition in which two or more variants of a specific genomic sequence can be found in a population. A “polymorphic site” is the locus at which the variation occurs. A single nucleotide polymorphism is a single base pair change. Typically a single nucleotide polymorphism is the replacement of one nucleotide by another nucleotide at the polymorphic site. Deletion of a single nucleotide or insertion of a single nucleotide, also give rise to single nucleotide polymorphisms. In the context of the present invention “single nucleotide polymorphism” preferably refers to a single nucleotide substitution. However, the polymorphism can also involve an insertion or a deletion of at least one nucleotide, preferably between 1 and 5 nucleotides. The nucleotide modification can also involve the presence of several adjacent single base polymorphisms. This type of nucleotide modification is usually called a “variable motif”. Generally, a “variable motif” involves the presence of 2 to 10 adjacent single base polymorphisms. In some instances, series of two or more single base polymorphisms can be interrupted by single bases which are not polymorphic. This is also globally considered to be a “variable motif”. Typically, between different genomes or between different individuals, the polymorphic site may be occupied by two different nucleotides.

[0062] The term “biallelic polymorphism” and “biallelic marker” are used interchangeably herein to refer to a single nucleotide polymorphism having two alleles at a fairly high frequency in the population. A “biallelic marker allele” refers to the nucleotide variants present at a biallelic marker site. Typically, the frequency of the less common allele of the biallelic markers of the present invention has been validated to be greater than 1%, preferably the frequency is greater than 10%, more preferably the frequency is at least 20% (i.e. heterozygosity rate of at least 0.32), even more preferably the frequency is at least 30% (i.e. heterozygosity rate of at least 0.42). A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker”.

[0063] The location of nucleotides in a polynucleotide with respect to the center of the polynucleotide are described herein in the following manner. When a polynucleotide has an odd number of nucleotides, the nucleotide at an equal distance from the 3′ and 5′ ends of the polynucleotide is considered to be “at the center” of the polynucleotide, and any nucleotide immediately adjacent to the nucleotide at the center, or the nucleotide at the center itself is considered to be “within 1 nucleotide of the center.” With an odd number of nucleotides in a polynucleotide any of the five nucleotides positions in the middle of the polynucleotide would be considered to be within 2 nucleotides of the center, and so on. When a polynucleotide has an even number of nucleotides, there would be a bond and not a nucleotide at the center of the polynucleotide. Thus, either of the two central nucleotides would be considered to be “within 1 nucleotide of the center” and any of the four nucleotides in the middle of the polynucleotide would be considered to be “within 2 nucleotides of the center”, and so on. For polymorphisms which involve the substitution, insertion or deletion of 1 or more nucleotides, the polymorphism, allele or biallelic marker is “at the center” of a polynucleotide if the difference between the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 3′ end of the polynucleotide, and the distance from the substituted, inserted, or deleted polynucleotides of the polymorphism and the 5′ end of the polynucleotide is zero or one nucleotide. If this difference is 0 to 3, then the polymorphism is considered to be “within 1 nucleotide of the center.” If the difference is 0 to 5, the polymorphism is considered to be “within 2 nucleotides of the center.” If the difference is 0 to 7, the polymorphism is considered to be “within 3 nucleotides of the center,” and so on.

[0064] As used herein the terminology “defining a biallelic marker” means that a sequence includes a polymorphic base from a biallelic marker. The sequences defining a biallelic marker may be of any length consistent with their intended use, provided that they contain a polymorphic base from a biallelic marker. The sequence is preferably between 1 and 500 nucleotides in length, more preferably between 5, 10, 15, 20, 25, or 40 and 200 nucleotides and still more preferably between 30 and 50 nucleotides in length. Each biallelic marker therefore corresponds to two forms of a polynucleotide sequence included in a gene, which, when compared with one another, present a nucleotide modification at one position. Preferably, the sequences defining a biallelic marker include a polymorphic base selected from the group consisting of biallelic markers A1 to A21. In some embodiments the sequences defining a biallelic marker comprise one of the sequences selected from the group consisting of SEQ ID Nos 30 to 71. Likewise, the term “marker” or “biallelic marker” requires that the sequence is of sufficient length to practically (although not necessarily unambiguously) identify the polymorphic allele, which usually implies a length of at least 4, 5, 6, 10, 15, 20, 25, or 40 nucleotides.

Variants And Fragments

[0065] 1. Polynucleotides

[0066] The invention also relates to variants and fragments of the polynucleotides described herein, particularly of a RBP-7 gene containing one or more biallelic markers according to the invention.

[0067] Variants of polynucleotides, as the term is used herein, are polynucleotides that differ from a reference polynucleotide. A variant of a polynucleotide may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally. Such non-naturally occurring variants of the polynucleotide may be made by mutagenesis techniques, including those applied to polynucleotides, cells or organisms. Generally, differences are limited so that the nucleotide sequences of the reference and the variant are closely similar overall and, in many regions, identical.

[0068] Variants of polynucleotides according to the invention include, without being limited to, nucleotide sequences that are at least 95% identical to any of SEQ ID Nos 1-28 or the sequences complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of any of SEQ ID Nos 1-28 or the sequences complementary thereto, and preferably at least 98% identical, more particularly at least 99.5% identical, and most preferably at least 99.9% identical to any of SEQ ID Nos 1-28 or the sequences complementary thereto or to any polynucleotide fragment of at least 8 consecutive nucleotides of any of SEQ ID Nos 1-28 or the sequences complementary thereto.

[0069] Changes in the nucleotide of a variant may be silent, which means that they do not alter the amino acids encoded by the polynucleotide.

[0070] However, nucleotide changes may also result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.

[0071] In the context of the present invention, particularly preferred embodiments are those in which the polynucleotides encode polypeptides which retain substantially the same biological function or activity as the mature RBP-7 protein.

[0072] A polynucleotide fragment is a polynucleotide having a sequence that entirely is the same as part but not all of a given nucleotide sequence, preferably the nucleotide sequence of a RBP-7 gene, and variants thereof. The fragment can be a portion of an exon or of an intron of a RBP-7 gene. It can also be a portion of the regulatory sequences of the RBP-7 gene. Preferably, such fragments comprise the polymorphic base of at least one of the biallelic markers of SEQ ID Nos. 30-71.

[0073] Such fragments may be “free-standing”, i.e. not part of or fused to other polynucleotides, or they may be comprised within a single larger polynucleotide of which they form a part or region. However, several fragments may be comprised within a single larger polynucleotide.

[0074] As representative examples of polynucleotide fragments of the invention, there may be mentioned those which are from about 4, 6, 8, 15, 20, 25, 40, 10 to 20, 10 to 30, 30 to 55, 50 to 100, 75 to 100 or 100 to 200 nucleotides in length. Preferred are those fragments which are about 47 nucleotides in length, such as those of SEQ ID Nos 30-71 or the sequences complementary thereto and containing at least one of the biallelic markers of a RBP-7 gene which are described herein. It will of course be understood that the polynucleotides of SEQ ID Nos 30-71 or the sequences complementary thereto can be shorter or longer, although it is preferred that they at least contain the polymorphic base of the biallelic marker which can be located at one end of the fragment or in the internal portion of the fragment.

[0075] 2. Polypeptides.

[0076] The invention also relates to variants, fragments, analogs and derivatives of the polypeptides described herein, including mutated RBP-7 proteins.

[0077] The variant may be 1) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more of the amino acid residues includes a substituent group, or 3) one in which the mutated RBP-7 is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or 4) one in which the additional amino acids are fused to the mutated RBP-7, such as a leader or secretory sequence or a sequence which is employed for purification of the mutated RBP-7 or a preprotein sequence. Such variants are deemed to be within the scope of those skilled in the art.

[0078] More particularly, a variant RBP-7 polypeptide comprises amino acid changes ranging from 1, 2, 3, 4, 5, 10 to 20 substitutions, additions or deletions of one amino acid, preferably from 1 to 10, more preferably from 1 to 5 and most preferably from 1 to 3 substitutions, additions or deletions of one amino acid. The preferred amino acid changes are those which have little or no influence on the biological activity or the capacity of the variant RBP-7 polypeptide to be recognized by antibodies raised against a native RBP-7 protein.

[0079] As illustrative embodiments of variant RBP-7 polypeptides encompassed by the present invention, there are the following polypeptides:

[0080] a polypeptide comprising a Glycine residue at the amino acid position 293 of the amino acid sequence of SEQ ID No. 29;

[0081] a polypeptide comprising a Glutamic acid at the amino acid in position 963 of SEQ ID No. 29; and,

[0082] a polypeptide comprising a Methionine residue at the amino acid position 969 of the amino acid sequence of SEQ ID No. 29.

[0083] By homologous peptide according to the present invention is meant a polypeptide containing one or several amino acid additions, deletions and/or substitutions in the amino acid sequence of a RBP-7 polypeptide. In the case of an amino acid substitution, one or several—consecutive or non-consecutive—amino acids are replaced by “equivalent” amino acids. The expression “equivalent” amino acid is used herein to designate any amino acid that may substituted for one of the amino acids belonging to the native protein structure without decreasing the binding properties of the corresponding peptides to the retinoblastoma proteins (i.e. RBP, p130, p107 etc.). In other words, the “equivalent” amino acids are those which allow the generation or the synthesis of a polypeptide with a modified sequence when compared to the amino acid sequence of the native RBP-7 protein, said modified polypeptide being able to bind to the retinoblastoma protein and/or to induce antibodies recognizing the parent polypeptide comprising, consisting essentially of, or consisting of a RBP-7 polypeptide.

[0084] These equivalent amino acids may be determined either by their structural homology with the initial amino acids to be replaced, by the similarity of their net charge, and optionally by the results of the cross-immunogenicity between the parent peptides and their modified counterparts.

[0085] By an equivalent amino acid according to the present invention is also meant the replacement of a residue in the L-form by a residue in the D form or the replacement of a Glutamic acid (E) residue by a Pyro-glutamic acid compound. The synthesis of peptides containing at least one residue in the D-form is, for example, described by Koch (Koch Y., 1977, Biochem. Biophys. Res. Commun., Vol. 74:488-491).

[0086] A specific, but not restrictive, embodiment of a modified peptide molecule of interest according to the present invention, which comprises, consists essentially of, or consists of a peptide molecule which is resistant to proteolysis, is a peptide in which the —CONH— peptide bond is modified and replaced by a (CH₂NH) reduced bond, a (NHCO) retro inverso bond, a (CH₂—O) methylene-oxy bond, a (CH₂—S) thiomethylene bond, a (CH₂CH₂) carba bond, a (CO—CH₂) cetomethylene bond, a (CHOH—CH₂) hydroxyethylene bond), a (N—N) bound, a E-alcene bond or also a —CH═CH— bond.

[0087] A polypeptide fragment is a polypeptide having a sequence that entirely is the same as part but not all of a given polypeptide sequence, preferably a polypeptide encoded by a RBP-7 gene and variants thereof. Preferred fragments include those regions possessing antigenic properties and which can be used to raise antibodies against the RBP-7 protein.

[0088] Such fragments may be “free-standing”, i.e. not part of or fused to other polypeptides, or they may be comprised within a single larger polypeptide of which they form a part or region. However, several fragments may be comprised within a single larger polypeptide.

[0089] As representative examples of polypeptide fragments of the invention, there may be mentioned those which comprise at least about 5, 6, 7, 8, 9 or 10 to 15, 10 to 20, 15 to 40, or 30 to 55 amino acids of the RBP-7 protein. In some embodiments, the fragments contain at least one amino acid mutation in the RBP-7 protein.

Complementary Polynucleotides

[0090] For the purpose of the present invention, a first polynucleotide is deemed to be complementary to a second polynucleotide when each base in the first polynucleotide is paired with its complementary base. Complementary bases are, generally, A and T (or A and U), or C and G.

Identity Between Nucleic Acids or Polypeptides

[0091] The terms “percentage of sequence identity” and “percentage homology” are used interchangeably herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. Homology is evaluated using any of the variety of sequence comparison algorithms and programs known in the art. Such algorithms and programs include, but are by no means limited to, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988; Altschul et al., 1990; Thompson et al., 1994; Higgins et al., 1996; Altschul et al., 1990; Altschul et al., 1993). In a particularly preferred embodiment, protein and nucleic acid sequence homologies are evaluated using the Basic Local Alignment Search Tool (“BLAST”) which is well known in the art (see, e.g., Karlin and Altschul, 1990; Altschul et al., 1990, 1993, 1997). In particular, five specific BLAST programs are used to perform the following task:

[0092] (1) BLASTP and BLAST3 compare an amino acid query sequence against a protein sequence database;

[0093] (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database;

[0094] (3) BLASTX compares the six-frame conceptual translation products of a query nucleotide sequence (both strands) against a protein sequence database;

[0095] (4) TBLASTN compares a query protein sequence against a nucleotide sequence database translated in all six reading frames (both strands); and

[0096] (5) TBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database.

[0097] The BLAST programs identify homologous sequences by identifying similar segments, which are referred to herein as “high-scoring segment pairs,” between a query amino or nucleic acid sequence and a test sequence which is preferably obtained from a protein or nucleic acid sequence database. High-scoring segment pairs are preferably identified (i.e., aligned) by means of a scoring matrix, many of which are known in the art. Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet et al., 1992; Henikoff and Henikoff, 1993). Less preferably, the PAM or PAM250 matrices may also be used (see, e.g., Schwartz and Dayhoff, eds., 1978). The BLAST programs evaluate the statistical significance of all high-scoring segment pairs identified, and preferably selects those segments which satisfy a user-specified threshold of significance, such as a user-specified percent homology. Preferably, the statistical significance of a high-scoring segment pair is evaluated using the statistical significance formula of Karlin (see, e.g., Karlin and Altschul, 1990). The programs listed above may be used with the default parameters or with modified parameters provided by the user.

RBP-7 Gene, Corresponding CDNAS and RBP-7 Coding and Regulatory Sequences

[0098] The gene encoding a RBP-7 polypeptide has been found by the inventors to be located on human chromosome 1, more precisely within the 1q43 locus of said chromosome. The RBP-7 gene has a length of about 166 kilobases and contains a 5′ regulatory region, 24 exons, and a 3′ regulatory region. A 5′-UTR region is spans the whole Exon 1 and the major portion of the 5′ end of Exon 2. A 3′-UTR region is spans the major portion of the 3′ end of Exon 24.

[0099] The present invention first concerns a purified or isolated nucleic acid encoding a Retinoblastoma Binding Protein named RBP-7 as well as a nucleic acid complementary thereto and fragments and variants thereof.

[0100] In particular, the invention concerns a purified or isolated nucleic acid comprising at least 8 consecutive nucleotides of a polynucleotide selected from the group consisting of SEQ ID Nos 1 and 4 as well as a nucleic acid sequence complementary thereto and fragments and variants thereof. The length of the fragments described above can range from at least 8, 10, 15, 20 or 30 to 200 nucleotides, preferably from at least 10 to 50 nucleotides, more preferably from at least 40 to 50 nucleotides. In some embodiments, the fragments may comprise more than 200 nucleotides of SEQ ID Nos. 1 and 4 or the sequences complementary thereto.

[0101] The invention also pertains to a purified or isolated nucleic acid of at least 8 nucleotides in length that hybridizes under stringent hybridization conditions with a polynucleotide selected from the group consisting of SEQ ID Nos 1 and 4 or the sequences complementary thereto. The length of the nucleic acids described above can range from 8, 10, 15, 20 or 30 to 200 nucleotides, preferably from 10 to 50 nucleotides, more preferably from 40 to 50 nucleotides. Such nucleic acids may be used as probes or primers, such as described in the corresponding section of the present specification.

[0102] The invention also encompasses a purified, isolated, or recombinant polynucleotide comprising a nucleotide sequence having at least 70, 75, 80, 85, 90, or 95% nucleotide identity with a nucleotide sequence of SEQ ID Nos 1 and 4 or a complementary sequence thereto or a fragment thereof. Percent identity may be determined using any of the programs and scoring matrices described above. For example, percent identity may be determined using BLASTN with the default parameters. In addition, the scoring matrix may be BLOSUM62

[0103] Particularly preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No. 1: 1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842-146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, 161453-162450. Additional preferred nucleic acids of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579. It should be noted that nucleic acid fragments of any size and sequence may also be comprised by the polynucleotides described in this section.

[0104] The main structural features of the RBP-7 gene are shown in FIG. 1. The upper line shows a structural map of the polynucleotide of SEQ ID No. 1 including the 24 exons, that are indicated by closed boxes, and the 23 introns, as well the 5′- and 3′-flanking regulatory regions. The position of the first nucleotide at 5′ end of each exon is also indicated, the nucleotide at position 1 being the first nucleotide at the 5′ end of the polynucleotide of SEQ ID No. 1.

[0105] Generally, an intron is defined as a nucleotide sequence that is present both in the genomic DNA and in the unspliced mRNA molecule, and which is absent from the mRNA molecule which has already gone through splicing events.

[0106] For the purpose of the present invention and in order to make a clear and unambiguous designation of the different nucleic acids encompassed, it has been postulated that the polynucleotides contained both in the nucleotide sequence of SEQ ID No. 1 and in the nucleotide sequences of SEQ ID No. 4 are considered as exonic sequences. Conversely, the polynucleotides contained in the nucleotide sequence of SEQ ID No. 1 and located between Exon 1 and 24, but which are absent both from the nucleotide sequence of SEQ ID No. 4 are considered as intronic sequences.

[0107] More precisely, the structural characteristics of the RBP-7 gene, as represented in FIG. 1 are as follows:

[0108] a) regulatory region, located between the nucleotide at position 1 and the nucleotide at position 273 of SEQ ID No. 1;

[0109] b) a “coding” region, located between the nucleotide at position 274 and the nucleotide at position 161451 of SEQ ID No. 1, comprising 24 exons and 23 introns, wherein said region defines the RBP-7 coding region.

[0110] c) a regulatory region, beginning at the nucleotide at position 161452 and ending at the nucleotide in position 162450 (the 3′-end nucleotide) of SEQ ID No. 1.

[0111] The translation start site ATG is located within the second exon and the translation stop codon is located within Exon 24 of the nucleotide sequence of SEQ ID No. 1.

[0112] The middle line of FIG. 1 shows the cDNA corresponding to the longest RBP-7 mRNA including the 24 exons. Each exon is represented by a specific box. The numbers located under the exon boxes indicate the nucleotide position of the 5′ end polynucleotide of each exon, it being understood that the nucleotide at position 1 is the 5′ end nucleotide of the cDNA. pAd denotes the four potential polyadenylation sites.

[0113] The lower line of FIG. 1 shows a map of the RBP-7 coding sequence (CDS), the start codon being located from the nucleotide in position 442 to the nucleotide in position 444 of the RBP-7 cDNA of SEQ ID No. 4 and the stop codon being located from the nucleotide in position 4378 to the nucleotide in position 4380 of the RBP-7 cDNA of SEQ ID No. 4.

[0114] The 24 exons included in the RBP-7 gene are represented in FIG. 1 and are described in Table A. TABLE A Beginning position End position Exon SEQ ID No. in SEQ ID No. 1 In SEQ ID No. 1 1 5 274 665 2 6 1466 1520 3 7 67593 67703 4 8 71119 71184 5 9 72599 72689 6 10 75544 75623 7 11 81842 81933 8 12 87902 88040 9 13 93857 93936 10 14 97159 97235 11 15 98963 99117 12 16 103570 103642 13 17 105085 105179 14 18 106683 106780 15 19 107799 108042 16 20 108376 108551 17 21 114336 114593 18 22 132247 132331 19 23 134151 134349 20 24 145566 146774 21 25 150447 150560 22 26 152960 153175 23 27 155591 155737 24 28 159702 161451

[0115] The middle line depicts the main structural features of a purified or isolated nucleic acid consisting longest cDNA that is obtained after reverse transcribing a mRNA generated after transcription of the RBP-7 gene. The longest mRNA has a nucleotide length of about 6 kilobases.

[0116] As it is depicted in FIG. 1, the main characteristics of the longest RBP-7 cDNA are the following:

[0117] a) A 5′-UTR region extending from the nucleotide at position 1 to the nucleotide at position 441 of SEQ ID No. 4;

[0118] b) An open reading frame (ORF) encoding the longest form of RBP-7 protein, wherein said ORF extends from the nucleotide at position 442 to the nucleotide at position 4380 of SEQ ID No. 4. The ATG translation start site is located between the nucleotide at position 442 and the nucleotide at position 444 of SEQ ID No. 4. The stop codon is located between the nucleotide at position 4378 and the nucleotide at position 4380 of SEQ ID No. 4.

[0119] c) A 3′-UTR region extending from the nucleotide at position 4381 to the nucleotide at position 6002 SEQ ID No. 4. This 3′-UTR region contains four potential polyadenylation sites comprises respectively the nucleotides between positions 4878 and 4883, 5116 and 5121, 5896 and between positions 5981 and 5986 of SEQ ID No. 4.

[0120]FIG. 2 is a representation of the RBP-7 gene in which the 24 exons are shown as closed boxes.

[0121] a) In each closed box that represents a given Exon, there are indicated both a number of base pairs corresponding to the non coding sequence eventually present in this Exon, and a number of amino acids. The number of amino acids is calculated as follows, starting from Exon 2: Exons 2 contains two complete codons and the first base of a third codon; only the two complete codons taken into account and the additional base is taken into account as the first base of the first codon of Exon 3, etc.;

[0122] b) The arrows above the Intron lines or above the Exon boxes indicate the localization of the different polymorphic markers of the invention on the RBP-7 gene, as well as their marker names;

[0123] c) The bold letters above exons 11 and 20 indicate the effect of the base changes constitutive to these polymorphic markers on the amino acid sequence of the resulting RBP-7 translation product.

[0124] The polynucleotide of SEQ ID No. 4 contains, from its 5′ end to its 3′ end, the sequences resulting from the 24 exons located in Table A on the RBP-7 genomic sequence, said exonic sequences being positioned on the RBP-7 cDNA of SEQ ID No. 4, as detailed in Table B below. TABLE B Beginning position End position Exon SEQ ID No. in SEQ ID No. 4 In SEQ ID No. 4 1 5 1 392 2 6 393 447 3 7 448 558 4 8 559 624 5 9 625 715 6 10 716 795 7 11 796 887 8 12 888 1026 9 13 1027 1106 10 14 1107 1183 11 15 1184 1338 12 16 1339 1411 13 17 1412 1507 14 18 1508 1604 15 19 1605 1848 16 20 1849 2024 17 21 2025 2282 18 22 2283 2367 19 23 2368 2566 20 24 2567 3775 21 25 3776 3889 22 26 3890 4105 23 27 4106 4252 24 28 4253 6002

[0125] The nucleotide sequence of the RBP-7 cDNA possesses some homologies with a cDNA encoding another human retinoblastoma binding protein, namely hRBP-1. This homology is randomly distributed throughout the whole cDNA sequences, without visible nucleic acid regions that are characteristic of conserved regions between cDNA sequences encoding different retinobastoma binding proteins.

[0126] The majority of interrupted genes are transcribed into a RNA that gives rise to a single type of spliced mRNA. But the RNAs of some genes follow patterns of alternative splicing, wherein a single gene gives rise to more than one mRNA species. In some cases, the ultimate pattern of expression is dictated by the primary transcript, because the use of different startpoints or termination sequences alters the splicing pattern. In other cases, a single primary transcript is spliced in more than one way, and internal exons are substituted, added or deleted. In some cases, the multiple products all are made in the same cell, but in others, the process is regulated so that particular splicing patterns occur only under particular conditions.

[0127] In the case of retinoblastoma binding proteins, alternative splicing patterns have been observed during the processing of the RBP1 pre-mRNA (Otterson et al., 1993). More precisely, alternative splicing of RBP1 clusters has been observed within a 207-nucleotide internal exon. From the four forms of mRNA detected, three of the predicted RBP1 peptides share amino-terminal and carboxy-terminal domains, while a fourth species encodes a distinct carboxy-terminal domain. Functional analysis of these peptides demonstrated that they are capable of precipitating retinoblastoma protein in vitro from K562 cell lysates, but cannot bind to mutant RB protein.

[0128] The inventors have found that a mRNA of about 6 kilobases and containing exon 1 of the RBP-7 gene at its 5′ end and exon 24 of the RBP-7 gene at its 3′ end, is produced in isolated cells from the prostate tissue, as described in Example 1.

[0129] Because the RBP-7 gene contains a large number of exons, it is expected that the corresponding pre-mRNA is processed in a family of mRNA molecules as a result of multiple alternative splicing events.

[0130] Additionally, individually combining each polynucleotide molecule defining a specific exon of the RBP-7 gene with at least one polynucleotide molecule defining another exon of the RBP-7 gene will give rise to a family of translation products that may be assayed for their biological functions of interaction with retinoblastoma proteins (i.e. pRb, p107, p130 etc.) or of interaction with DNA sequences of the type recognized by the transcription factors of the E2F family. Such translation products have a shorter size than that of the resulting protein encoded by the longest RBP-7 mRNA and thus may be advantageously used in therapeutics, as compared with the longest polypeptides, due to their weaker immunogenicity, for example.

[0131] Consequently, a further aspect of the present invention is a purified or isolated nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID Nos 5-28 or the sequences complementary thereto.

[0132] The invention also deals with a purified or isolated nucleic acid comprising a combination of at least two polynucleotides selected from the group consisting of SEQ ID Nos 5-28 or the sequences complementary thereto, wherein the polynucleotides are ordered within the nucleic acid, from the 5′ end to the 3′ end of said nucleic acid, in the same order as in the SEQ ID No. 1.

[0133] In this specific embodiment of a purified or isolated nucleic acid according to the invention, said nucleic acid preferably comprises SEQ ID Nos 5 and 6 at its 5′ end and SEQ ID No. 28 at its 3′ end.

Regulatory Regions

[0134] As already mentioned hereinbefore, the polynucleotide of SEQ ID No. 1 contains regulatory regions both in the non-coding 5′-flanking region (SEQ ID No. 2) and the non-coding 3′-flanking region (SEQ ID No. 3) that border the coding sequences.

[0135] The promoter activity of the regulatory region contained in SEQ ID No. 1 can be assessed as described below.

[0136] Genomic sequences lying upstream of the RBP-7 gene are cloned into a suitable promoter reporter vector, such as the pSEAP-Basic, pSEAP-Enhancer, pβgal-Basic, pβgal-Enhancer, or pEGFP-1 Promoter Reporter vectors available from Clontech. Briefly, each of these promoter reporter vectors include multiple cloning sites positioned upstream of a reporter gene encoding a readily assayable protein such as secreted alkaline phosphatase, β0 galactosidase, or green fluorescent protein. The sequences upstream of the RBP-7 coding region are inserted into the cloning sites upstream of the reporter gene in both orientations and introduced into an appropriate host cell. The level of reporter protein is assayed and compared to the level obtained from a vector which lacks an insert in the cloning site. The presence of an elevated expression level in the vector containing the insert with respect to the control vector indicates the presence of a promoter in the insert. If necessary, the upstream sequences can be cloned into vectors which contain an enhancer for increasing transcription levels from weak promoter sequences. A significant level of expression above that observed with the vector lacking an insert indicates that a promoter sequence is present in the inserted upstream sequence.

[0137] Promoter sequences within the upstream genomic DNA may be further defined by constructing nested deletions in the upstream DNA using conventional techniques such as Exonuclease III digestion. The resulting deletion fragments can be inserted into the promoter reporter vector to determine whether the deletion has reduced or obliterated promoter activity. In this way, the boundaries of the promoters may be defined. If desired, potential individual regulatory sites within the promoter may be identified using site directed mutagenesis or linker scanning to obliterate potential transcription factor binding sites within the promoter, individually or in combination. The effects of these mutations on transcription levels may be determined by inserting the mutations into the cloning sites in the promoter reporter vectors.

[0138] Polynucleotides carrying the regulatory elements located both at the 5′ end and at the 3′ end of the RBP-7 coding region may be advantageously used to control the transcriptional and translational activity of an heterologous polynucleotide of interest.

[0139] A 5′ regulatory polynucleotide of the invention may include the 5′-untranslated region (5′-UTR) or the sequence complementary thereto, or a biologically active fragment or variant thereof. The 5′-regulatory polynucleotide harbors a CAAT box from the nucleotide in position 139 to the nucleotide in position 147 of the nucleotide sequence of SEQ ID No. 2. Additionally, the 5′-regulatory polynucleotide of the invention comprises a TATA box from the nucleotide in position 199 to the nucleotide in position 205 of the nucleotide sequence of SEQ ID No. 2.

[0140] A 3′ regulatory polynucleotide of the invention may include the 3′-untranslated region (3′-UTR) or the sequences complementary thereto, or a biologically active fragment or variant thereof.

[0141] Another aspect of the present invention is a purified and/or isolated polynucleotide located at the 5′ end of the start codon of the RBP-7 gene, wherein said polynucleotide carries expression and/or regulation signals allowing the expression of the RBP-7 gene. Thus, another part of the present invention is a purified or isolated nucleic acid comprising a nucleotide sequence of SEQ ID No. 2 and functionally active fragments or variants thereof. The fragments may be of any length to facilitate the expression and/or regulation of a gene operably linked thereto. In particular, the fragments may contain one or more binding sites for transcription factors. In some embodiments, the fragments at least 8, 10, 15, 20 or 30 to 200 nucleotides of SEQ ID No. 2. In other embodiments, the fragments may comprise more than 200 nucleotides of SEQ ID No. 2 or the sequence complementary thereto.

[0142] The invention further deals with a purified and/or isolated polynucleotide located at the 3′ end of the stop codon of the RBP-7 gene, wherein said polynucleotide carries regulation signals involved in the expression of the RBP-7 gene. Thus another part of the present invention is a purified or isolated nucleic acid comprising a nucleotide sequence of SEQ ID No. 3, the sequence complementary thereto, and functionally active fragments or variants thereof. The fragments may be of any length to facilitate the expression and/or regulation of a gene operationally linked thereto. In some embodiments, the fragments may comprise at least 8, 10, 15, 20 or 30 to 200 nucleotides of SEQ ID No. 3 or the sequence complementary thereto. In other embodiments, the fragments may comprise more than 200 nucleotides of SEQ ID No. 3 or the sequence complementary thereto.

[0143] Thus, the invention also pertains to a purified or isolated nucleic acid which is selected from the group consisting of:

[0144] a) a nucleic acid comprising the nucleotide sequence SEQ ID No. 2 or the sequence complementary thereto;

[0145] b) a nucleic acid comprising a biologically active fragment or variant of the nucleic acid of SEQ ID No. 2 or the sequence complementary thereto.

[0146] In a specific embodiment of the above nucleic acid, said nucleic acid includes the 5′-untranslated region (5′-UTR) located between the nucleotide at position 1 to the nucleotide at position 441 of SEQ ID No. 4, or the sequences complementary thereto, or a biologically active fragment or variant thereof.

[0147] Another aspect of the present invention is a purified or isolated nucleic acid which is selected from the group consisting of:

[0148] a) a nucleic acid comprising the nucleotide sequence SEQ ID No. 3 or the sequence complementary thereto;

[0149] b) a nucleic acid comprising a biologically active fragment, a variant of the nucleic acid of SEQ ID No. 3 or the sequence complementary thereto.

[0150] In a specific embodiment of the above nucleic acid, said nucleic acid includes the 3′-untranslated region (3′-UTR) located between the nucleotide at position 4381 and the nucleotide at position 6002 of SEQ ID No. 4, or the sequences complementary thereto, or a biologically active fragment or variant thereof.

[0151] Preferred fragments of the nucleic acid of SEQ ID No. 2 or the sequence complementary thereto have a range of length from 100, 125, 150, 175, 200 to 225, 250, 273 consecutive nucleotides. Preferred fragments will comprise both the CAAT box and the TATA box of the nucleotide sequence of SEQ ID No. 2.

[0152] Preferred fragments of the nucleic acid of SEQ ID No. 3 or the sequence complementary thereto have a length of about 600 nucleotides, more particularly of about 300 nucleotides, more preferably of about 200 nucleotides and most preferably about 100 nucleotides.

[0153] In order to identify the relevant biologically active polynucleotide derivatives of SEQ ID No. 3, one may follow the procedures described in Sambrook et al. (1989, the disclosure of which is incorporated herein by reference) relating to the use of a recombinant vector carrying a marker gene (i.e. β galactosidase, chloramphenicol acetyl transferase, etc.) the expression of which will be detected when placed under the control of a biologically active derivative polynucleotide of SEQ ID No. 3.

[0154] Regulatory polynucleotides of the invention may be prepared from the nucleotide sequence of SEQ ID No. 1 or the sequences complementary thereto by cleavage using the suitable restriction enzymes, as described in Sambrook et al. (1989), supra.

[0155] Regulatory polynucleotides may also be prepared by digestion of the nucleotide sequence of SEQ ID No. 1 or the sequences complementary thereto by an exonuciease enzyme, such as Bal31 (Wabiko et al., 1986).

[0156] These regulatory polynucleotides can also be prepared by nucleic acid chemical synthesis, as described elsewhere in the specification, when oligonucleotide probes or primers synthesis is disclosed.

[0157] The regulatory polynucleotides according to the invention may advantageously be part of a recombinant expression vector that may be used to express a coding sequence in a desired host cell or host organism. The recombinant expression vectors according to the invention are described elsewhere in the specification.

[0158] The above defined polynucleotides that carry the expression and/or regulation signals of the RBP-7 gene may be used, for example as part of a recombinant vector, in order to drive the expression of a desired polynucleotide, said desired polynucleotide being either (1) a polynucleotide encoding a RBP-7 protein, or a fragment or variant thereof, or (2) an “heterologous” polynucleotide, such as a polynucleotide encoding a desired “heterologous” polypeptide or a desired RNA in a recombinant cell host.

[0159] The invention also encompasses a polynucleotide comprising, consisting essentially of, or consisting of:

[0160] a) a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 2, or the sequence complementary thereto, or a biologically active fragment or variant thereof;

[0161] b) a polynucleotide encoding a desired polypeptide or nucleic acid.

[0162] c) Optionally, a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 3, or the sequence complementary thereto, or a biologically active fragment or variant thereof.

[0163] In a preferred embodiment, a polynucleotide such as disclosed above comprises the nucleic acid of SEQ ID No. 2, or the sequences complementary thereto, or a fragment, a variant or a biologically active derivative thereof which is located at the 5′ end of the polynucleotide encoding the desired polypeptide or polynucleotide.

[0164] In another embodiment, a polynucleotide such as that above described comprises the nucleic acid of SEQ ID No. 3, or the sequence complementary thereto, or a fragment, a variant or a biologically active derivative thereof which is located at the 3′ end of the polynucleotide encoding the desired polypeptide or nucleic acid. A preferred desired nucleic acid comprises of a ribonucleic acid useful as antisense molecule.

[0165] The desired polypeptide encoded by the above described nucleic acid may be of various nature or origin, encompassing proteins of prokaryotic or eukaryotic origin. Among the polypeptides which may be expressed under the control of a RBP-7 regulatory region are bacterial, fungal or viral antigens. Are also encompassed eukaryotic proteins such as intracellular proteins, such as “house keeping” proteins, membrane-bound proteins, such as receptors, and secreted proteins such as the numerous endogenous mediators including cytokines.

[0166] The desired nucleic acid encoded by the above described polynucleotide, usually a RNA molecule, may be complementary to a RBP-7 coding sequence and thus useful as an antisense polynucleotide.

[0167] Such a polynucleotide may be included in a recombinant expression vector in order to express a desired polypeptide or a desired polynucleotide in host cell or in a host organism. Suitable recombinant vectors that contain a polynucleotide such as described hereinbefore are disclosed elsewhere in the specification.

Coding Regions

[0168] As depicted in FIG. 1, the RBP-7 open reading frame is contained in the longest RBP-7 which mRNA has a nucleotide length of about 4 kilobases.

[0169] More precisely, the effective RBP-7 coding sequence (CDS) is between the nucleotide at position 442 and the nucleotide at position 4377 of SEQ ID No. 4.

[0170] The invention further provides a purified or isolated nucleic acid comprising a polynucleotide selected from the group consisting of a polynucleotide comprising a nucleic acid sequence located between the nucleotide at position 442 and the nucleotide at position 4377 of SEQ ID No. 4, or the sequence complementary thereto, or a variant or fragment thereof or a sequence complementary thereto.

[0171] A further object of the present invention comprises polynucleotide fragments of the RBP-7 gene that are useful for the detection of the presence of an unaltered or an altered copy of the RBP-7 gene within the genome of a host organism and also for the detection and/or quantification of the expression of the RBP-7 gene in said host organism.

[0172] Thus, another object of the present invention is a purified or isolated nucleic acid encoding a variant or a mutated RBP-7 protein.

[0173] A first preferred embodiment of a copy of the RBP-7 gene comprises an allele in which a single base substitution in the codon encoding the Aspartic acid (D) residue in amino acid position 293 of the RBP-7 protein of SEQ ID No. 29 leads to the amino acid replacement for a Glycine (G) residue.

[0174] A second preferred embodiment of a copy of the RBP-7 gene comprises an allele in which a single base substitution in the codon encoding the Glycine (G) residue in amino acid position 963 of the RBP-7 protein of SEQ ID No. 29 leads to the amino acid replacement for a Glutamic acid (E) residue.

[0175] A third preferred embodiment of a copy of the RBP-7 gene comprises an allele in which a single base substitution in the codon encoding the Leucine (L) residue in amino acid position 969 of the RBP-7 protein of SEQ ID No. 29 leads to the amino acid replacement for a Methionine (M) residue.

[0176] Thus, another object of the present invention is a purified or isolated nucleic acid encoding a mutated RBP-7 protein.

[0177] The above disclosed polynucleotide that contains only coding sequences derived from the RBP-7 ORF may be expressed in a desired host cell or a desired host organism, when said polynucleotide is placed under the control of suitable expression signals. Such a polynucleotide, when placed under the suitable expression signals, may be inserted in a vector for its expression.

Oligonucleotide Probes and Primers

[0178] Polynucleotides derived from the RBP-7 gene described above are useful in order to detect the presence of at least a copy of a nucleotide sequence of SEQ ID No. 1, or a fragment or a variant thereof in a test sample.

[0179] The present invention concerns a purified or isolated nucleic acid comprising at least 8 consecutive nucleotides of the nucleotide sequence SEQ ID No. 1 or a sequence complementary thereto or variants thereof. In another embodiment, the present invention relates to nucleic acids comprising at least 8, 10, 15, 20 or 30 to 200 nucleotides, preferably from at least 10 to 50 nucleotides, more preferably from at least 40 to 50 nucleotides of SEQ ID No. 1 or the sequence complementary thereto. In some embodiments, the nucleic acids may comprise more than 200 nucleotides of SEQ ID No. 1 or the sequence complementary thereto.

[0180] Particularly preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 1 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No. 1: 1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842-146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, 161453-162450.

[0181] The invention also relates to an oligonucleotide of at least at least 8 nucleotides in length that hybridizes under stringent hybridization conditions with a nucleic acid selected from the group consisting of the nucleotide sequences 1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842-146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, 161453-162450 of SEQ ID No. 1 or a variant thereof or a sequence complementary thereto. In some embodiments, the invention relates to sequences comprising at least 8, 10, 15, 20 or 30 to 200 nucleotides, preferably from at least 10 to 50 nucleotides, more preferably from 40 to 50 nucleotides of SEQ ID No. 1 or the sequence complementary thereto or variants thereof. In some embodiments, the invention relates to sequences comprising more than 200 nucleotides of SEQ ID No. 1 or the sequence complementary thereto.

[0182] For the purpose of defining such a hybridizing nucleic acid according to the invention, the stringent hybridization conditions are the following:

[0183] the hybridization step is realized at 65° C. in the presence of 6×SSC buffer, 5×Denhardt's solution, 0,5% SDS and 100 μg/ml of salmon sperm DNA.

[0184] The hybridization step is followed by four washing steps:

[0185] two 5 min washings, preferably at 65° C. in a 2×SSC and 0.1% SDS buffer;

[0186] one 30 min washing, preferably at 65° C. in a 2×SSC and 0.1% SDS buffer,

[0187] one 10 min washing, preferably at 65° C. in a 0.1×SSC and 0.1%SDS buffer,

[0188] the above hybridization conditions are suitable for a nucleic acid molecule of about 20 nucleotides in length. There is no need to say that the hybridization conditions described above can readily be adapted according to the length of the desired nucleic acid, following techniques well known to the one skilled in the art. The hybridization conditions may for example be adapted according to the teachings disclosed in the book of Hames and Higgins (1985), the disclosure of which is incorporated herein by reference.

[0189] Another aspect of the invention is a purified or isolated nucleic acid comprising at least 8 consecutive nucleotides of the nucleotide sequence SEQ ID No. 4 or the sequence complementary thereto or variants thereof. In another embodiment, the nucleic acid comprises from at least 8, 10, 15, 20 or 30 to 200 nucleotides, preferably from at least 10 to 50 nucleotides, more preferably from at least 40 to 50 nucleotides of SEQ ID No. 4 or the sequence complementary thereto or variants thereof. In some embodiments, the fragments may comprise more than 200 nucleotides of SEQ ID No. 4 or the sequence complementary thereto or variants thereof.

[0190] Additional preferred probes and primers of the invention include isolated, purified, or recombinant polynucleotides comprising a contiguous span of at least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150, 200, 500, or 1000 nucleotides of SEQ ID No. 4 or the complements thereof, wherein said contiguous span comprises at least 1, 2, 3, 5, or 10 of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579.

[0191] Alternatively, the invention also relates to an oligonucleotide of at least 8 nucleotides in length that hybridizes under the stringent hybridization conditions previously defined with a nucleic acid selected from the group consisting of the nucleotide sequences 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579 of SEQ ID No. 1 or a variant thereof or a sequence complementary thereto.

[0192] A nucleic probe or primer according to the invention comprises at least 8 consecutive nucleotides of a polynucleotide of SEQ ID Nos 1 or 4 or the sequences complementary thereto, preferably from 8 to 200 consecutive nucleotides, more particularly from 10, 15, 20 or 30 to 100 consecutive nucleotides, more preferably from 10 to 50 nucleotides, and most preferably from 40 to 50 consecutive nucleotides of a polynucleotide of SEQ ID Nos 1 or 4 or the sequences complementary thereto.

[0193] In a first preferred embodiment, the probe or primer is suspended in a suitable buffer for performing a hybridization or an amplification reaction.

[0194] In a second embodiment, the oligonucleotide probe, which may be immobilized on a support, is capable of hybridizing with a RBP-7 gene, preferably with a region of the RBP-7 gene which comprises a biallelic marker of the present invention. The techniques for immobilizing a nucleotide primer or probe on a solid support are well-known to the skilled artisan and include, but are not limited to, the immobilization techniques described in the present application.

[0195] In a third embodiment, the primer is complementary to any nucleotide sequence of the RBP-7 gene and can be used to amplify a region of the RBP-7 gene contained in the nucleic acid sample to be tested which includes a polymorphic base of at least one biallelic marker. Preferably, the amplified region includes a polymorphic base of at least one biallelic marker selected from the group consisting of SEQ ID Nos 30-71 or the sequences complementary thereto. In some embodiments, the primer comprises one of the sequences of SEQ ID Nos 72-101 and 102-136.

[0196] When using a polynucleotide probe or primer in a detection method of the invention, the DNA or RNA contained in the sample to be assayed may be subjected to a first extraction step well known to the one skilled in the art, in order to make the DNA or RNA material contained in the initial sample available to a hybridization reaction, prior to the hybridization step itself.

[0197] The nucleic acid probes and primers of the invention are also used to detect and/or amplify a portion of the RBP-7 gene within which a polymorphism or a mutation causes a change either in the expression level of the RBP-7 gene or a change in the amino acid sequence of the RBP-7 gene translation product.

[0198] The invention further concerns detection or amplification kits containing a pair of oligonucleotide primers or an oligonucleotide probe according to the invention. The kits of the present invention can also comprise optional elements including appropriate amplification reagents such as DNA polymerases when the kit comprises primers, or reagents useful in hybridization between a labeled hybridization probe and a RBP-7 gene containing at least one biallelic marker. In one embodiment, the biallelic marker comprises one of the sequences of SEQ ID Nos 30-71 or the sequences complementary thereto.

[0199] In one embodiment the invention encompasses isolated, purified, and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos 1 and 4 and the complement thereof, wherein said span includes a biallelic marker of RBP-7 in said sequence; optionally, wherein said biallelic marker of RBP-7 is selected from the group consisting of A1 to A2 1, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein said contiguous span is 18 to 47 nucleotides in length and said biallelic marker is within 4 nucleotides of the center of said polynucleotide; optionally, wherein said polynucleotide consists of or comprises said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide; optionally, wherein the 3′ end of said contiguous span is present at the 3′ end of said polynucleotide; and optionally, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide and said biallelic marker is present at the 3′ end of said polynucleotide. In a preferred embodiment, said probes comprises, consists of, or consists essentially of a sequence selected from the sequences SEQ ID Nos 30-71 and the complementary sequences thereto.

[0200] In another embodiment the invention encompasses isolated, purified and recombinant polynucleotides comprising, consisting of, or consisting essentially of a contiguous span of 8 to 50 nucleotides of SEQ ID Nos 1 and 4 or the complements thereof, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide, and wherein the 3′ end of said polynucleotide is located within 20 nucleotides upstream of a biallelic marker of RBP-7 in said sequence; optionally, wherein said biallelic marker of RBP-7 is selected from the group consisting of A1 to A21, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith; optionally, wherein the 3′ end of said polynucleotide is located 1 nucleotide upstream of said biallelic marker of RBP-7 in said sequence; and optionally, wherein said polynucleotide comprises, consists of, or consists essentially of a sequence selected from the sequences SEQ ID Nos 102-136.

[0201] In a further embodiment, the invention encompasses isolated, purified, or recombinant polynucleotides comprising, consisting of, or consisting essentially of a sequence selected from the sequences SEQ ID Nos 72-101.

[0202] In an additional embodiment, the invention encompasses polynucleotides for use in hybridization assays, sequencing assays, and enzyme-based mismatch detection assays for determining the identity of the nucleotide at a biallelic marker of RBP-7 in SEQ ID Nos 1 and 4, or the complements thereof, as well as polynucleotides for use in amplifying segments of nucleotides comprising a biallelic marker of RBP-7 in SEQ ID Nos 1 and 4, or the complements thereof, optionally, wherein said biallelic marker of RBP-7 is selected from the group consisting of A1 to A21, and the complements thereof, or optionally the biallelic markers in linkage disequilibrium therewith.

[0203] The formation of stable hybrids depends on the melting temperature (Tm) of the DNA. The Tm depends on the length of the primer or probe, the ionic strength of the solution and the G+C content. The higher the G+C content of the primer or probe, the higher is the melting temperature because G:C pairs are held by three H bonds whereas A:T pairs have only two. The GC content in the probes and primers of the invention usually ranges between 10 and 75%, preferably between 35 and 60%, and more preferably between 40 and 55%.

[0204] The length of these probes and probes can range from 8, 10, 15, 20, or 30 to 100 nucleotides, preferably from 10 to 50, more preferably from 15 to 30 nucleotides. Shorter probes and primers tend to lack specificity for a target nucleic acid sequence and generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. Longer probes and primers are expensive to produce and can sometimes self-hybridize to form hairpin structures. The appropriate length for primers and probes under a particular set of assay conditions may be empirically determined by one of skill in the art.

[0205] The primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis by a method such as the phosphodiester method of Narang et al. (1979), the phosphodiester method of Brown et al. (1979), the diethylphosphoramidite method of Beaucage et al. (1981) and the solid support method described in EP 0 707 592, the disclosures of which are incorporated herein by reference in their entireties.

[0206] Any of the polynucleotides of the present invention can be labeled, if desired, by incorporating a label detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive substances (³²p, ³⁵s, ³H, ¹²⁵I), fluorescent dyes (5-bromodesoxyuridin, fluorescein, acetylaminofluorene, digoxigenin) or biotin. Preferably, polynucleotides are labeled at their 3′ and 5′ ends. Examples of non-radioactive labeling of nucleic acid fragments are described in the French Patent No. FR-7810975 or by Urdea et al (1988) or Sanchez-Pescador et al (1988). Advantageously, the probes according to the present invention may have structural characteristics such that they allow the signal amplification, such structural characteristics being, for example, branched DNA probes as those described by Urdea et al. in 1991 or in the European Patent No. EP-0225,807, the disclosure of which is incorporated herein by reference in its entirety (Chiron).

[0207] A label can also be used to capture the primer, so as to facilitate the immobilization of either the primer or a primer extension product, such as amplified DNA, on a solid support. A capture label is attached to the primers or probes and can be a specific binding member which forms a binding pair with the solid's phase reagent's specific binding member (e.g. biotin and streptavidin). Therefore depending upon the type of label carried by a polynucleotide or a probe, it may be employed to capture or to detect the target DNA. Further, it will be understood that the polynucleotides, primers or probes provided herein, may, themselves, serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of a primer or probe to thereby immobilize the primer or probe to the solid phase. In cases where a polynucleotide probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or “tail” that is not complementary to the target. In the case where a polynucleotide primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase. DNA Labeling techniques are well known to the skilled technician.

[0208] The probes of the present invention are useful for a number of purposes. They can be notably used in Southern hybridization to genomic DNA. The probes can also be used to detect PCR amplification products. They may also be used to detect mismatches in the RBP-7 gene or mRNA using other techniques.

[0209] Any of the polynucleotides, primers and probes of the present invention can be conveniently immobilized on a solid support. Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, sheep (or other animal) red blood cells, duracytes and others. The solid support is not critical and can be selected by one skilled in the art. Thus, latex particles, microparticles, magnetic or non-magnetic beads, membranes, plastic tubes, walls of microtliter wells, glass or silicon chips, sheep (or other suitable animal's) red blood cells and duracytes are all suitable examples. Suitable methods for immobilizing nucleic acids on solid phases include ionic, hydrophobic, covalent interactions and the like. A solid support, as used herein, refers to any material which is insoluble, or can be made insoluble by a subsequent reaction. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid phase can retain an additional receptor which has the ability to attract and immobilize the capture reagent. The additional receptor can include a charged substance that is oppositely charged with respect to the capture reagent itself or to a charged substance conjugated to the capture reagent. As yet another alternative, the receptor molecule can be any specific binding member which is immobilized upon (attached to) the solid support and which has the ability to immobilize the capture reagent through a specific binding reaction. The receptor molecule enables the indirect binding of the capture reagent to a solid support material before the performance of the assay or during the performance of the assay. The solid phase thus can be a plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cells, duracytes® and other configurations known to those of ordinary skill in the art. The polynucleotides of the invention can be attached to or immobilized on a solid support individually or in groups of at least 2, 5, 8, 10, 12, 15, 20, or 25 distinct polynucleotides of the inventions to a single solid support. In addition, polynucleotides other than those of the invention may attached to the same solid support as one or more polynucleotides of the invention.

[0210] Consequently, the invention also deals with a method for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos 1, 4, a fragment or a variant thereof or the complementary sequence thereto in a sample, said method comprising the following steps of:

[0211] a) bringing into contact a nucleic acid probe or a plurality of nucleic acid probes as described above and the sample to be assayed.

[0212] b) detecting the hybrid complex formed between the probe and a nucleic acid in the sample.

[0213] In a first preferred embodiment of this detection method, said nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule.

[0214] In a second preferred embodiment of said method, said nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate.

[0215] The invention further concerns a kit for detecting the presence of a nucleic acid comprising a nucleotide sequence selected from a group consisting of SEQ ID Nos 1, 4, a fragment or a variant thereof or the complementary sequence thereto in a sample, said kit comprising:

[0216] a) a nucleic acid probe or a plurality of nucleic acid probes as described above;

[0217] b) optionally, the reagents necessary for performing the hybridization reaction.

[0218] In a first preferred embodiment of the detection kit, the nucleic acid probe or the plurality of nucleic acid probes are labeled with a detectable molecule.

[0219] In a second preferred embodiment of the detection kit, the nucleic acid probe or the plurality of nucleic acid probes has been immobilized on a substrate.

Oligonucleotide Arrays

[0220] A substrate comprising a plurality of oligonucleotide primers or probes of the invention may be used either for detecting or amplifying targeted sequences in the RBP-7 gene and may also be used for detecting mutations in the coding or in the non-coding sequences of the RBP-7 gene.

[0221] Any polynucleotide provided herein may be attached in overlapping areas or at random locations on the solid support. Alternatively the polynucleotides of the invention may be attached in an ordered array wherein each polynucleotide is attached to a distinct region of the solid support which does not overlap with the attachment site of any other polynucleotide. Preferably, such an ordered array of polynucleotides is designed to be “addressable” where the distinct locations are recorded and can be accessed as part of an assay procedure. Addressable polynucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. The knowledge of the precise location of each polynucleotides location makes these “addressable” arrays particularly useful in hybridization assays. Any addressable array technology known in the art can be employed with the polynucleotides of the invention. One particular embodiment of these polynucleotide arrays is known as the Genechips™, and has been generally described in U.S. Pat. No. 5,143,854; PCT publications WO 90/15070 and 92/10092, the disclosures of which are incorporated herein by reference in their entireties. These arrays may generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis (Fodor et al., Science, 251:767-777, 1991). The immobilization of arrays of oligonucleotides on solid supports has been rendered possible by the development of a technology generally identified as “Very Large Scale Immobilized Polymer Synthesis” (VLSIPS™) in which, typically, probes are immobilized in a high density array on a solid surface of a chip. Examples of VLSRPS™ technologies are provided in U.S. Pat. Nos. 5,143,854 and 5,412,087 and in PCT Publications WO 90/15070, WO 92/10092 and WO 95/11995, the disclosures of which are incorporated herein by reference in their entireties, which describe methods for forming oligonucleotide arrays through techniques such as light-directed synthesis techniques. In designing strategies aimed at providing arrays of nucleotides immobilized on solid supports, further presentation strategies were developed to order and display the oligonucleotide arrays on the chips in an attempt to maximize hybridization patterns and sequence information. Examples of such presentation strategies are disclosed in PCT Publications WO 94/12305, WO 94/11530, WO 97/29212 and WO 97/31256, the disclosures of which are incorporated herein by reference in their entireties.

[0222] In another embodiment of the oligonucleotide arrays of the invention, an oligonucleotide probe matrix may advantageously be used to detect mutations occurring in the RBP-7 gene and in its regulatory region. For this particular purpose, probes are specifically designed to have a nucleotide sequence allowing their hybridization to the genes that carry known mutations (either by deletion, insertion of substitution of one or several nucleotides). By known mutations is meant mutations on the RBP-7 gene that have been identified according, for example to the technique used by Huang et al. (1996) or Samson et al. (1996).

[0223] Another technique that is used to detect mutations in the RBP-7 gene is the use of a high-density DNA array. Each oligonucleotide probe constituting a unit element of the high density DNA array is designed to match a specific subsequence of the RBP-7 genomic DNA or cDNA. Thus, an array comprising, consisting essentially of, or consisting of oligonucleotides complementary to subsequences of the target gene sequence is used to determine the identity of the target sequence with the wild gene sequence, measure its amount, and detect differences between the target sequence and the reference wild gene sequence of the RBP-7 gene. One such design, termed 4L tiled array, uses a set of four probes (A, C, G, T), preferably 15-nucleotide oligomers. In each set of four probes, the perfect complement will hybridize more strongly than mismatched probes. Consequently, a nucleic acid target of length L is scanned for mutations with a tiled array containing 4L probes, the whole probe set containing all the possible mutations in the known wild reference sequence. The hybridization signals of the 15-mer probe set tiled array are perturbed by a single base change in the target sequence. As a consequence, there is a characteristic loss of signal or a “footprint” for the probes flanking a mutation position. This technique was described by Chee et al. in 1996, which is herein incorporated by reference.

[0224] Consequently, the invention concerns an array of nucleic acid comprising at least one polynucleotide described above as probes and primers. Preferably, the invention concerns an array of nucleic acid comprising at least two polynucleotides described above as probes and primers.

Amplification of the RBP-7 GENE

[0225] 1. DNA Extraction

[0226] As for the source of the genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the context of the present invention is from peripheral venous blood of each donor.

[0227] The techniques of DNA extraction are well-known to the skilled technician. Such techniques are described notably by Lin et al. (1998) and by Mackey et al. (1998).

[0228] 2. DNA Amplification

[0229] DNA amplification techniques are well-known to those skilled in the art. Amplification techniques that can be used in the context of the present invention include, but are not limited to, the ligase chain reaction (LCR) described in EP-A-320 308, WO 9320227 and EP-A-439 182, the disclosures of which are incorporated herein by reference, the polymerase chain reaction (PCR, RT-PCR) and techniques such as the nucleic acid sequence based amplification (NASBA) described in Guatelli J C, et al. (1990) and in Compton J. (1991), Q-beta amplification as described in European Patent Application No. 4544610, strand displacement amplification as described in Walker et al. (1996) and EP A 684 315 and, target mediated amplification as described in PCT Publication WO 9322461, the disclosure of which is incorporated herein by reference.

[0230] LCR and Gap LCR are exponential amplification techniques, both depend on DNA ligase to join adjacent primers annealed to a DNA molecule. In Ligase Chain Reaction (LCR), probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to target. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5′ phosphate-3′ hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Of course, if the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes, which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. A method for multiplex LCR has also been described (WO 9320227). Gap LCR (GLCR) is a version of LCR where the probes are not adjacent but are separated by 2 to 3 bases.

[0231] For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770 or, to use Asymmetric Gap LCR (RT-AGLCR) as described by Marshall et al. (1994). AGLCR is a modification of GLCR that allows the amplification of RNA.

[0232] The PCR technology is the preferred amplification technique used in the present invention. A variety of PCR techniques are familiar to those skilled in the art. For a review of PCR technology, see White (1997) and the publication entitled “PCR Methods and Applications” (1991, Cold Spring Harbor Laboratory Press). In each of these PCR procedures, PCR primers on either side of the nucleic acid sequences to be amplified are added to a suitably prepared nucleic acid sample along with dNTPs and a thermostable polymerase such as Taq polymerase, Pfu polymerase, or Vent polymerase. The nucleic acid in the sample is denatured and the PCR primers are specifically hybridized to complementary nucleic acid sequences in the sample. The hybridized primers are extended. Thereafter, another cycle of denaturation, hybridization, and extension is initiated. The cycles are repeated multiple times to produce an amplified fragment containing the nucleic acid sequence between the primer sites. PCR has further been described in several patents including U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,965,188. Each of these publications is incorporated by reference.

[0233] One of the aspects of the present invention is a method for the amplification of the human RBP-7 gene, particularly of the genomic sequences of SEQ ID No. 1 or of the cDNA sequence of SEQ ID No. 4, or a fragment or a variant thereof in a test sample, preferably using the PCR technology. The method comprises the steps of contacting a test sample suspected of containing the target RBP-7 encoding sequence or portion thereof with amplification reaction reagents comprising a pair of amplification primers, and eventually in some instances a detection probe that can hybridize with an internal region of amplicon sequences to confirm that the desired amplification reaction has taken place.

[0234] Thus, the present invention also relates to a method for the amplification of a human RBP-7 gene sequence, particularly of a portion of the genomic sequences of SEQ ID) No. 1 or of the cDNA sequence of SEQ ID No. 4, or a variant thereof in a test sample, said method comprising the steps of:

[0235] a) contacting a test sample suspected of containing the targeted RBP-7 gene sequence comprised in a nucleotide sequence selected from a group consisting of SEQ ID Nos 1 and 4, or fragments or variants thereof with amplification reaction reagents comprising a pair of amplification primers as described above and located on either side of the polynucleotide region to be amplified, and

[0236] b) optionally detecting the amplification products.

[0237] In a preferred embodiment of the above amplification method, the amplification product is detected by hybridization with a labeled probe having a sequence which is complementary to the amplified region.

[0238] The primers are more particularly characterized in that they have sufficient complementarity with any sequence of a strand of the genomic sequence close to the region to be amplified, for example with a non-coding sequence adjacent to exons to amplify.

[0239] In a particular embodiment of the invention, the primers are selected form the group consisting of the nucleotide sequences detailed in Table C below. TABLE C Position range of Complementary position Forward Primer amplification primer in Reverse Primer range of amplification Name SEQ ID No. 1 Name primer in SEQ ID No. 1 P1  313-330 P26 732-751 P2  1282-1299 P27 1682-1699 P3  67531-67549 P28 67810-67830 P4  70927-70945 P29 71257-71276 P5  71613-71631 P30 72043-72060 P6  75390-75409 P31 75795-75814 P7  77544-77563 P32 77926-77943 P8  81708-81726 P33 82108-82127 P9  105046-105065 P34 105326-105345 P10 104751-104770 P35 105297-105316 P11 107691-107710 P36 108091-108110 P12 114296-114315 P37 114698-114716 P13 114327-114345 P38 114735-114753 P14 132101-132118 P39 132504-132521 P15 145522-145541 P40 145923-145942 P16 145866-145884 P41 146266-146285 P17 145956-145976 P42 146399-146418 P18 146529-146547 P43 146955-146972 P19 152763-152780 P44 153164-153182 P20 155404-155422 P45 155706-155726 P21 160043-160060 P46 160445-160462 P22 160361-160378 P47 160770-160788 P23 160742-160759 P48 161147-161165 P24 161127-161144 P49 161530-161547 P25 161217-161235 P50 161617-161636

[0240] The invention also concerns a kit for the amplification of a human RBP-7 gene sequence, particularly of a portion of the genomic sequences of SEQ ID No. 1 or of the cDNA sequence of SEQ ID No. 4, or a variant thereof in a test sample, wherein said kit comprises:

[0241] a) A pair of oligonucleotide primers located on either side of the RBP-7 region to be amplified;

[0242] b) Optionally, the reagents necessary for performing the amplification reaction.

[0243] In a preferred embodiment of the amplification kit described above, the primers are selected from the group consisting of the nucleotide sequences of SEQ ID Nos 72-101 and P1-P50.

[0244] In another embodiment of the above amplification kit, the amplification product is detected by hybridization with a labeled probe having a sequence which is complementary to the amplified region.

Biallelic Markers of RBP-7

[0245] The inventors have discovered nucleotide polymorphisms located within the genomic DNA containing the RBP-7 gene, and among them “Single Nucleotide Polymorphisms” or SNPs that are also termed biallelic markers.

[0246] The invention also relates to a nucleotide sequence, preferably a purified and/or isolated polynucleotide comprising a sequence defining a biallelic marker located in the sequence of a RBP-7 gene, a fragment or variant thereof or a sequence complementary thereto. The sequences defining a biallelic marker may be of any length consistent with their intended use, provided that they contain a polymorphic base from a biallelic marker. Preferably, the sequences defining a biallelic marker include the polymorphic base of one of SEQ ID Nos 30-71 or the sequence complementary thereto. In some embodiments the sequences defining a biallelic marker comprise one of the sequences selected from the group consisting of SEQ ID Nos 30-71 or the sequences complementary thereto.

[0247] In a preferred embodiment, the invention relates to a set of purified and/or isolated nucleotide sequences, each sequence comprising a sequence defining a biallelic marker located in the sequence of a RBP-7 gene, wherein the set is characterized in that between about 30 and 100%, preferably between about 40 and 60%, more preferably between 50 and 60%, of the sequences defining a biallelic marker are selected from the group consisting of SEQ ID Nos 30-71, the sequences complementary thereto, or a fragment or variant thereof.

[0248] The invention further concerns a nucleic acid encoding a RBP-7 protein, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos 30-71 or the sequences complementary thereto.

[0249] The invention also relates to nucleotide sequence selected from the group consisting of SEQ ID Nos 30-71, the sequences complementary thereto, or a fragment or a variant thereof.

[0250] A) Identification of Biallelic Markers

[0251] There are two preferred methods through which the biallelic markers of the present invention can be generated.

[0252] In a first method, DNA samples from unrelated individuals are pooled together, following which the genomic DNA of interest is amplified and sequenced. The nucleotide sequences thus obtained are then analyzed to identify significant polymorphisms. One of the major advantages of this method resides in the fact that the pooling of the DNA samples substantially reduces the number of DNA amplification reactions and sequencing reactions which must be carried out. Moreover, this method is sufficiently sensitive so that a biallelic marker obtained therewith usually shows a sufficient degree of informativeness for conducting association studies.

[0253] In a second method for generating biallelic markers, the DNA samples are not pooled and are therefore amplified and sequenced individually. The resulting nucleotide sequences obtained are then also analyzed to identify significant polymorphisms.

[0254] It will readily be appreciated that when this second method is used, a substantially higher number of DNA amplification reactions and sequencing reactions must be carried out. Moreover, a biallelic marker obtained using this method may show a lower degree of informativeness for conducting association studies, e.g. if the frequency of its less frequent allele may be less than about 10%. It will further be appreciated that including such less informative biallelic markers in association studies to identify potential genetic associations with a trait may allow in some cases the direct identification of causal mutations, which may, depending on their penetrance, be rare mutations. This method is usually preferred when biallelic markers need to be identified in order to perform association studies within candidate genes.

[0255] The following is a description of the various parameters of a preferred method used by the inventors to generate the markers of the present invention.

[0256] 1-DNA Extraction

[0257] The genomic DNA samples from which the biallelic markers of the present invention are generated are preferably obtained from unrelated individuals corresponding to a heterogeneous population of known ethnic background.

[0258] The term “individual” as used herein refers to vertebrates, particularly members of the mammalian species and includes but is not limited to domestic animals, sports animals, laboratory animals, primates and humans. Preferably, the individual is a human.

[0259] The number of individuals from whom DNA samples are obtained can vary substantially, preferably from about 10 to about 1000, preferably from about 50 to about 200 individuals. It is usually preferred to collect DNA samples from at least about 100 individuals in order to have sufficient polymorphic diversity in a given population to identify as many markers as possible and to generate statistically significant results.

[0260] As for the source of the genomic DNA to be subjected to analysis, any test sample can be foreseen without any particular limitation. These test samples include biological samples which can be tested by the methods of the present invention described herein and include human and animal body fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts, tears, saliva, milk, white blood cells, myelomas and the like; biological fluids such as cell culture supernatants; fixed tissue specimens including tumor and non-tumor tissue and lymph node tissues; bone marrow aspirates and fixed cell specimens. The preferred source of genomic DNA used in the context of the present invention is from peripheral venous blood of each donor.

[0261] The techniques of DNA extraction are well-known to the skilled technician. Details of a preferred embodiment are provided in Example 2.

[0262] Once genomic DNA from every individual in the given population has been extracted, it is preferred that a fraction of each DNA sample is separated, after which a pool of DNA is constituted by assembling equivalent amounts of the separated fractions into a single one. However, the person skilled in the art can choose to amplify the pooled or unpooled sequences

[0263] 2—DNA Amplification

[0264] The identification of biallelic markers in a sample of genomic DNA may be facilitated through the use of DNA amplification methods. DNA samples can be pooled or unpooled for the amplification step. DNA amplification techniques are well known to those skilled in the art. Various methods to amplify DNA fragments carrying biallelic markers are further described hereinbefore in “Amplification of the RBP-7 gene”. The PCR technology is the preferred amplification technique used to identify new biallelic markers. A typical example of a PCR reaction suitable for the purposes of the present invention is provided in Example 3.

[0265] In this context, one of the groups of oligonucleotides according to the present invention is a group of primers useful for the amplification of a genomic sequence encoding RBP-7. The primers pairs are characterized in that they have sufficient complementarity with any sequence of a strand of the RBP-7 gene to be amplified, preferably with a sequence of introns adjacent to exons to amplify, with regions of the 3′ and 5′ ends of the RBP-7 gene, with splice sites or with 5′ UTRs or 3′ UTRs to hybridize therewith.

[0266] These primers focus on exons and splice sites of the RBP-7 gene since an identified biallelic marker as described below presents a higher probability to be an eventual causal mutation if it is located in these functional regions of the gene.

[0267] 15 pairs of primers were designed with the aim of amplifying each of the 24 exons of the RBP-7 gene (Table 1). To these primers can be added, at either end thereof, a further polynucleotide useful for sequencing such as described in Example 3. Preferred primers include those having the nucleotide sequences disclosed in Example 3. Some of the primers according to the invention allow the amplification of the majority of the RBP-7 Exons shown in FIG. 2.

[0268] The primers described above are individually useful as oligonucleotide probes in order to detect the corresponding RBP-7 nucleotide sequence in a sample, and more preferably to detect the presence of a RBP-7 DNA or RNA molecule in a sample suspected to contain it.

[0269] 3—Sequencing of Amplified Genomic DNA and Identification of Polymorphisms

[0270] The amplification products generated as described above with the primers of the invention are then sequenced using methods known and available to the skilled technician. Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol.

[0271] Following gel image analysis and DNA sequence extraction, sequence data are automatically processed with adequate software to assess sequence quality

[0272] The sequence data obtained as described above are transferred to a database, where quality control and validation steps are performed. A base-caller, working using a Unix system automatically flags suspect peaks, taking into account the shape of the peaks, the inter-peak resolution, and the noise level. The base-caller also performs an automatic trimming. Any stretch of 25 or fewer bases having more than 4 suspect peaks is usually considered unreliable and is discarded.

[0273] After this first sequence quality analysis, polymorphism analysis software is used to detect the presence of biallelic sites among individual or pooled amplified fragment sequences. The polymorphism search is based on the presence of superimposed peaks in the electrophoresis pattern. These peaks, which present two distinct colors, correspond to two different nucleotides at the same position on the sequence. In order for peaks to be considered significant, peak height has to satisfy conditions of ratio between the peaks and conditions of ratio between a given peak and the surrounding peaks of the same color.

[0274] However, since the presence of two peaks can be an artifact due to background noise, two controls are utilized to exclude these artifacts:

[0275] the two DNA strands are sequenced and a comparison between the peaks is carried out. The polymorphism has to be detected on both strands for validation.

[0276] all the sequencing electrophoresis patterns of the same amplification product provided from distinct pools and/or individuals are compared. The homogeneity and the ratio of homozygous and heterozygous peak height are controlled through these distinct DNAs.

[0277] The detection limit for the frequency of biallelic polymorphisms detected by sequencing pools of 100 individuals is about 0.1 for the minor allele, as verified by sequencing pools of known allelic frequencies. However, more than 90% of the biallelic polymorphisms detected by the pooling method have a frequency for the minor allele higher than 0.25. Therefore, the biallelic markers selected by this method have a frequency of at least 0.1 for the minor allele and less than 0.9 for the major allele, preferably at least 0.2 for the minor allele and less than 0.8 for the major allele, more preferably at least 0.3 for the minor allele and less than 0.7 for the major allele, thus a heterozygosity rate higher than 0.18, preferably higher than 0.32, more preferably higher than 0.42.

[0278] In a particular embodiment of the invention, the test samples are a pool of 100 individuals and 50 individual samples. This is the methodology used in the preferred embodiment of the present invention, in which 21 biallelic markers have been identified in a genomic region containing the RBP-7 gene. Their location on the genomic RBP-7 DNA is shown in FIG. 2 and their particular sequences are disclosed in example 4. The 24 exons and the intronic sequences surrounding the exons were analyzed. Among the 21 biallelic markers identified within the RBP-7 gene, 6 biallelic markers are located within 4 different exons, and 15 biallelic markers are located within the different intronic regions. The biallelic markers 5-130-257, 5-143-84 and 5-143-101 respectively change asparagine into glycine, glycine into glutamic acid and leucine into methionine in the RBP-7 protein. The amino acid changes caused by the 5-143-84 biallelic marker may be important for the RBP-7 biological activity, since a neutral amino acid is replaced by a positively charged amino acid in a RBP-7 region likely to contain a domain involved in a non-covalent interaction with the retinoblastoma protein or also a pRb related protein such as p107 or p130.

[0279] 4—Validation of the Biallelic Markers of the Present Invention

[0280] The polymorphisms are evaluated for their usefulness as genetic markers by validating that both alleles are present in a population. Validation of the biallelic markers is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. Microsequencing is a preferred method of genotyping alleles. The validation by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group can be as small as one individual if that individual is heterozygous for the allele in question. Preferably the group contains at least three individuals, more preferably the group contains five or six individuals, so that a single validation test will be more likely to result in the validation of more of the biallelic markers that are being tested. It should be noted, however, that when the validation test is performed on a small group it may result in a false negative result if as a result of sampling error none of the individuals tested carries one of the two alleles. Thus, the validation process is less useful in demonstrating that a particular initial result is an artifact, than it is at demonstrating that there is a bona fide biallelic marker at a particular position in a sequence. All of the genotyping, haplotyping, and association study methods of the invention may optionally be performed solely with validated biallelic markers.

[0281] 5—Evaluation of the Frequency of the Biallelic Markers of the Present Invention

[0282] The validated biallelic markers are further evaluated for their usefulness as genetic markers by determining the frequency of the least common allele at the biallelic marker site. The higher the frequency of the less common allele the greater the usefulness of the biallelic marker in association and interaction studies. The determination of the least common allele is accomplished by genotyping a group of individuals by a method of the invention and demonstrating that both alleles are present. This determination of frequency by genotyping step may be performed on individual samples derived from each individual in the group or by genotyping a pooled sample derived from more than one individual. The group must be large enough to be representative of the population as a whole. Preferably the group contains at least 20 individuals, more preferably the group contains at least 50 individuals, most preferably the group contains at least 100 individuals. Of course the larger the group the greater the accuracy of the frequency determination because of reduced sampling error. A biallelic marker wherein the frequency of the less common allele is 30% or more is termed a “high quality biallelic marker.” All of the genotyping, haplotyping, and association interaction study methods of the invention may optionally be performed solely with high quality biallelic markers.

[0283] B—Genotyping an Individual for Biallelic Markers

[0284] Methods are provided to genotype a biological sample for one or more biallelic markers of the present invention, all of which may be performed in vitro. Such methods of genotyping comprise determining the identity of a nucleotide at an RBP-7 biallelic marker site by any method known in the art. These methods find use in genotyping case-control populations in association studies as well as individuals in the context of detection of alleles of biallelic markers which are known to be associated with a given trait, in which case both copies of the biallelic marker present in individual's genome are determined so that an individual may be classified as homozygous or heterozygous for a particular allele.

[0285] These genotyping methods can be performed nucleic acid samples derived from a single individual or pooled DNA samples.

[0286] Genotyping can be performed using similar methods as those described above for the identification of the biallelic markers, or using other genotyping methods such as those further described below. In preferred embodiments, the comparison of sequences of amplified genomic fragments from different individuals is used to identify new biallelic markers whereas microsequencing is used for genotyping known biallelic markers in diagnostic and association study applications.

[0287] 1—Source Of DNA For Genotyping

[0288] Any source of nucleic acids, in purified or non-purified form, can be utilized as the starting nucleic acid, provided it contains or is suspected of containing the specific nucleic acid sequence desired. DNA or RNA may be extracted from cells, tissues, body fluids and the like as described above in “DNA extraction”. While nucleic acids for use in the genotyping methods of the invention can be derived from any mammalian source, the test subjects and individuals from which nucleic acid samples are taken are generally understood to be human.

[0289] 2—Amplification of DNA Fragments Comprising Biallelic Markers

[0290] Methods and polynucleotides are provided to amplify a segment of nucleotides comprising one or more biallelic marker of the present invention. It will be appreciated that amplification of DNA fragments comprising biallelic markers may be used in various methods and for various purposes and is not restricted to genotyping. Nevertheless, many genotyping methods, although not all, require the previous amplification of the DNA region carrying the biallelic marker of interest. Such methods specifically increase the concentration or total number of sequences that span the biallelic marker or include that site and sequences located either distal or proximal to it. Diagnostic assays may also rely on amplification of DNA segments carrying a biallelic marker of the present invention.

[0291] Amplification of DNA may be achieved by any method known in the art. Amplification techniques are described above under the headings “Amplification of the RBP-7 gene”.

[0292] Some of these amplification methods are particularly suited for the detection of single nucleotide polymorphisms and allow the simultaneous amplification of a target sequence and the identification of the polymorphic nucleotide as it is further described below.

[0293] The identification of biallelic markers as described above allows the design of appropriate oligonucleotides, which can be used as primers to amplify DNA fragments comprising the biallelic markers of the present invention. Amplification can be performed using the primers initially used to discover new biallelic markers which are described herein or any set of primers allowing the amplification of a DNA fragment comprising a biallelic marker of the present invention.

[0294] In some embodiments the present invention provides primers for amplifying a DNA fragment containing one or more biallelic markers of the present invention. Preferred amplification primers are listed in Example 3. It will be appreciated that the primers listed are merely exemplary and that any other set of primers which produce amplification products containing one or more biallelic markers of the present invention.

[0295] The spacing of the primers determines the length of the segment to be amplified. In the context of the present invention amplified segments carrying biallelic markers can range in size from at least about 25 bp to 35 kbp. Amplification fragments from 25-3000 bp are typical, fragments from 50-1000 bp are preferred and fragments from 100-600 bp are highly preferred. It will be appreciated that amplification primers for the biallelic markers may be any sequence which allow the specific amplification of any DNA fragment carrying the markers. Amplification primers may be labeled or immobilized on a solid support as described under the headings entitled “Oligonucleotide probes and primers”.

[0296] 3—Methods of Genotyping DNA Samples for Biallelic Markers

[0297] a—Sequencing Assays

[0298] The amplification products generated above with the primers of the invention can be sequenced using methods known and available to the skilled technician. Preferably, the amplified DNA is subjected to automated dideoxy terminator sequencing reactions using a dye-primer cycle sequencing protocol. A sequence analysis can allow the identification of the base present at the polymorphic site.

[0299] b—Microsequencing Assays

[0300] In microsequencing methods, the nucleotide at a polymorphic site in a target DNA is detected by a single nucleotide primer extension reaction. This method involves appropriate microsequencing primers which, hybridize just upstream of the polymorphic base of interest in the target nucleic acid. A polymerase is used to specifically extend the 3′ end of the primer with one single ddNTP (chain terminator) complementary to the nucleotide at the polymorphic site. Next the identity of the incorporated nucleotide is determined in any suitable way.

[0301] Typically, microsequencing reactions are carried out using fluorescent ddNTPs and the extended microsequencing primers are analyzed by electrophoresis on ABI 377 sequencing machines to determine the identity of the incorporated nucleotide as described in EP 412 883, the disclosure of which is incorporated herein by reference in its entirety. Alternatively capillary electrophoresis can be used in order to process a higher number of assays simultaneously. An example of a typical microsequencing procedure that can be used in the context of the present invention is provided in Example 5.

[0302] Different approaches can be used for the labeling and detection of ddNTPs. A homogeneous phase detection method based on fluorescence resonance energy transfer has been described by Chen and Kwok (1997) and Chen et al. (1997). In this method amplified genomic DNA fragments containing polymorphic sites are incubated with a 5′-fluorescein-labeled primer in the presence of allelic dye-labeled dideoxyribonucleoside triphosphates and a modified Taq polymerase. The dye-labeled primer is extended one base by the dye-terminator specific for the allele present on the template. At the end of the genotyping reaction, the fluorescence intensities of the two dyes in the reaction mixture are analyzed directly without separation or purification. All these steps can be performed in the same tube and the fluorescence changes can be monitored in real time.

[0303] Microsequencing may be achieved by the established microsequencing method or by developments or derivatives thereof. Alternative methods include several solid-phase microsequencing techniques. The basic microsequencing protocol is the same as described previously, except that the method is conducted as a heterogenous phase assay, in which the primer or the target molecule is immobilized or captured onto a solid support. To simplify the primer separation and the terminal nucleotide addition analysis, oligonucleotides are attached to solid supports or are modified in such ways that permit affinity separation as well as polymerase extension. The 5′ ends and internal nucleotides of synthetic oligonucleotides can be modified in a number of different ways to permit different affinity separation approaches, e.g., biotinylation. If a single affinity group is used on the oligonucleotides, the oligonucleotides can be separated from the incorporated terminator regent. This eliminates the need of physical or size separation. More than one oligonucleotide can be separated from the terminator reagent and analyzed simultaneously if more than one affinity group is used. This permits the analysis of several nucleic acid species or more nucleic acid sequence information per extension reaction. The affinity group need not be on the priming oligonucleotide but could alternatively be present on the template. For example, immobilization can be carried out via an interaction between biotinylated DNA and streptavidin-coated microtitration wells or avidin-coated polystyrene particles. In the same manner oligonucleotides or templates may be attached to a solid support in a high-density format. In such solid phase microsequencing reactions, incorporated ddNTPs can be radiolabeled (Syvänen, 1994) or linked to fluorescein (Livak and Hainer, 1994). The detection of radiolabeled ddNTPs can be achieved through scintillation-based techniques. The detection of fluorescein-linked ddNTPs can be based on the binding of antifluorescein antibody conjugated with alkaline phosphatase, followed by incubation with a chromogenic substrate (such as p-nitrophenyl phosphate). Other possible reporter-detection pairs include: ddNTP linked to dinitrophenyl (DNP) and anti-DNP alkaline phosphatase conjugate (Harju et al., 1993) or biotinylated ddNTP and horseradish peroxidase-conjugated streptavidin with o-phenylenediamine as a substrate (WO 92/15712, the disclosure of which is incorporated herein by reference in its entirety). As yet another alternative solid-phase microsequencing procedure, Nyren et al. (1993) described a method relying on the detection of DNA polymerase activity by an enzymatic luminometric inorganic pyrophosphate detection assay (ELIDA).

[0304] Pastinen et al. (1997) describe a method for multiplex detection of single nucleotide polymorphism in which the solid phase minisequencing principle is applied to an oligonucleotide array format. High-density arrays of DNA probes attached to a solid support (DNA chips) are further described below.

[0305] In one aspect the present invention provides polynucleotides and methods to genotype one or more biallelic markers of the present invention by performing a microsequencing assay. Preferred microsequencing primers include those being featured in Example 5. It will be appreciated that the microsequencing primers listed in Example 5 are merely exemplary and that, any primer having a 3′ end immediately adjacent to the polymorphic nucleotide may be used. Similarly, it will be appreciated that microsequencing analysis may be performed for any biallelic marker or any combination of biallelic markers of the present invention. One aspect of the present invention is a solid support which includes one or more microsequencing primers listed in Example 5, or fragments comprising at least 8, at least 12, at least 15, or at least 20 consecutive nucleotides thereof and having a 3′ terminus immediately upstream of the corresponding biallelic marker, for determining the identity of a nucleotide at a biallelic marker site.

c—Mismatch Detection Assays Based on Polymerases and Ligases

[0306] In one aspect the present invention provides polynucleotides and methods to determine the allele of one or more biallelic markers of the present invention in a biological sample, by allele-specific amplification assays. Methods, primers and various parameters to amplify DNA fragments comprising biallelic markers of the present invention are further described above.

Allele Specific Amplification Primers

[0307] Discrimination between the two alleles of a biallelic marker can also be achieved by allele specific amplification, a selective strategy, whereby one of the alleles is amplified without amplification of the other allele. This can be accomplished by placing the polymorphic base at the 3′ end of one of the amplification primers. Because the extension forms from the 3′ end of the primer, a mismatch at or near this position has an inhibitory effect on amplification. Therefore, under appropriate amplification conditions, these primers only direct amplification on their complementary allele. Determining the precise location of the mismatch and the corresponding assay conditions are well within the ordinary skill in the art.

Ligation/Amplification Based Methods

[0308] The “Oligonucleotide Ligation Assay” (OLA) uses two oligonucleotides which are designed to be capable of hybridizing to abutting sequences of a single strand of a target molecules. One of the oligonucleotides is biotinylated, and the other is detectably labeled. If the precise complementary sequence is found in a target molecule, the oligonucleotides will hybridize such that their termini abut, and create a ligation substrate that can be captured and detected. OLA is capable of detecting single nucleotide polymorphisms and may be advantageously combined with PCR as described by Nickerson et al. (1990). In this method, PCR is used to achieve the exponential amplification of target DNA, which is then detected using OLA.

[0309] Other amplification methods which are particularly suited for the detection of single nucleotide polymorphism include LCR (ligase chain reaction), Gap LCR (GLCR) which are described above in “Amplification of the RBP-7 gene”. LCR uses two pairs of probes to exponentially amplify a specific target. The sequences of each pair of oligonucleotides, is selected to permit the pair to hybridize to abutting sequences of the same strand of the target. Such hybridization forms a substrate for a template-dependant ligase. In accordance with the present invention, LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a biallelic marker site. In one embodiment, either oligonucleotide will be designed to include the biallelic marker site. In such an embodiment, the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the biallelic marker on the oligonucleotide. In an alternative embodiment, the oligonucleotides will not include the biallelic marker, such that when they hybridize to the target molecule, a “gap” is created as described in WO 90/01069, the disclosure of which is incorporated herein by reference in its entirety. This gap is then “filled” with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential allele-specific amplification of the desired sequence is obtained.

[0310] Ligase/Polymerase-mediated Genetic Bit Analysis™ is another method for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule (WO 95/21271, the disclosure of which is incorporated herein by reference in its entirety). This method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide. The reaction is monitored by detecting a specific label attached to the reaction's solid phase or by detection in solution.

d—Hybridization Assay Methods

[0311] A preferred method of determining the identity of the nucleotide present at a biallelic marker site involves nucleic acid hybridization. The hybridization probes, which can be conveniently used in such reactions, preferably include the probes defined herein. Any hybridization assay may be used including Southern hybridization, Northern hybridization, dot blot hybridization and solid-phase hybridization (see Sambrook et al., 1989).

[0312] Hybridization refers to the formation of a duplex structure by two single stranded nucleic acids due to complementary base pairing. Hybridization can occur between exactly complementary nucleic acid strands or between nucleic acid strands that contain minor regions of mismatch. Specific probes can be designed that hybridize to one form of a biallelic marker and not to the other and therefore are able to discriminate between different allelic forms. Allele-specific probes are often used in pairs, one member of a pair showing perfect match to a target sequence containing the original allele and the other showing a perfect match to the target sequence containing the alternative allele. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Stringent, sequence specific hybridization conditions, under which a probe will hybridize only to the exactly complementary target sequence are well known in the art (Sambrook et al., 1989). Stringent conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. Although such hybridizations can be performed in solution, it is preferred to employ a solid-phase hybridization assay. The target DNA comprising a biallelic marker of the present invention may be amplified prior to the hybridization reaction. The presence of a specific allele in the sample is determined by detecting the presence or the absence of stable hybrid duplexes formed between the probe and the target DNA. The detection of hybrid duplexes can be carried out by a number of methods. Various detection assay formats are well known which utilize detectable labels bound to either the target or the probe to enable detection of the hybrid duplexes. Typically, hybridization duplexes are separated from unhybridized nucleic acids and the labels bound to the duplexes are then detected. Those skilled in the art will recognize that wash steps may be employed to wash away excess target DNA or probe as well as unbound conjugate. Further, standard heterogeneous assay formats are suitable for detecting the hybrids using the labels present on the primers and probes. Preferably, the hybrids can be bound to a solid phase reagent by virtue of a capture label and detected by virtue of a detection label. In cases where the detection label is directly detectable, the presence of the hybrids on the solid phase can be detected by causing the label to produce a detectable signal, if necessary, and detecting the signal. In cases where the label is not directly detectable, the captured hybrids can be contacted with a conjugate, which generally comprises a binding member attached to a directly detectable label. The conjugate becomes bound to the complexes and the conjugates presence on the complexes can be detected with the directly detectable label. Thus, the presence of the hybrids on the solid phase reagent can be determined.

[0313] The polynucleotides provided herein can be used to produce probes which can be used in hybridization assays for the detection of biallelic marker alleles in biological samples. These probes are characterized in that they preferably comprise between 8 and 50 nucleotides, and in that they are sufficiently complementary to a sequence comprising a biallelic marker of the present invention to hybridize thereto and preferably sufficiently specific to be able to discriminate the targeted sequence for only one nucleotide variation. A particularly preferred probe is 25 nucleotides in length. Preferably the polymorphic site of the biallelic marker is within 4 nucleotides of the center of the polynucleotide probe. In particularly preferred probes the polymorphic site of the biallelic marker is at the center of said polynucleotide.

[0314] Preferably the probes of the present invention are labeled or immobilized on a solid support. Labels and solid supports are further described in “Oligonucleotide probes and primers”. Detection probes are generally nucleic acid sequences or uncharged nucleic acid analogs such as, for example peptide nucleic acids which are disclosed in International Patent Application WO 92/20702, morpholino analogs which are described in U.S. Pat. Nos. 5,185,444; 5,034,506 and 5,142,047, the disclosures of which are incorporated herein by reference in their entireties. The probe may have to be rendered “non-extendable” in that additional dNTPs cannot be added to the probe. In and of themselves analogs usually are non-extendable and nucleic acid probes can be rendered non-extendable by modifying the 3′ end of the probe such that the hydroxyl group is No. longer capable of participating in elongation. For example, the 3′ end of the probe can be functionalized with the capture or detection label to thereby consume or otherwise block the hydroxyl group. Alternatively, the 3′ hydroxyl group simply can be cleaved, replaced or modified, U.S. patent application Ser. No. 07/049,061 filed Apr. 19, 1993 describes modifications, which can be used to render a probe non-extendable.

[0315] The probes of the present invention are useful for a number of purposes. By assaying the hybridization to an allele specific probe, one can detect the presence or absence of a biallelic marker allele in a given sample.

[0316] High-Throughput parallel hybridizations in array format are specifically encompassed within “hybridization assays” and are described below.

[0317] e—Hybridization to Addressable Arrays of Oligonucleotides

[0318] DNA chips result from the adaptation of computer chips to biology. Efficient access to polymorphism information is obtained through a basic structure comprising high-density arrays of oligonucleotide probes attached to a solid support (the chip) at selected positions. Each DNA chip can contain thousands to millions of individual synthetic DNA probes arranged in a grid-like pattern and miniaturized to the size of a dime.

[0319] The chip technology has already been applied with success in numerous cases. For example, the screening of mutations has been undertaken in the BRCA1 gene, in S. cerevisiae mutant strains, and in the protease gene of HIV-1 virus (Hacia et al., 1996; Shoemaker et al., 1996 ; Kozal et al., 1996). Chips of various formats for use in detecting biallelic polymorphisms can be produced on a customized basis by Affymetrix (GeneChip™), Hyseq (HyChip and HyGnostics), and Protogene Laboratories.

[0320] In general, these methods employ arrays of oligonucleotide probes that are complementary to target nucleic acid sequence segments from an individual which, target sequences include a polymorphic marker. EP785280, the disclosure of which is incorporated herein by reference in its entirety, describes a tiling strategy for the detection of single nucleotide polymorphisms. Briefly, arrays may generally be “tiled” for a large number of specific polymorphisms. By “tiling” is generally meant the synthesis of a defined set of oligonucleotide probes which is made up of a sequence complementary to the target sequence of interest, as well as preselected variations of that sequence, e.g., substitution of one or more given positions with one or more members of the basis set of monomers, i.e. nucleotides. Tiling strategies are further described in PCT Application No. WO 95/11995, the disclosure of which is incorporated herein by reference in its entirety. In a particular aspect, arrays are tiled for a number of specific, identified biallelic marker sequences. In particular the array is tiled to include a number of detection blocks, each detection block being specific for a specific biallelic marker or a set of biallelic markers. For example, a detection block may be tiled to include a number of probes, which span the sequence segment that includes a specific polymorphism. To ensure probes that are complementary to each allele, the probes are synthesized in pairs differing at the biallelic marker. In addition to the probes differing at the polymorphic base, monosubstituted probes are also generally tiled within the detection block. These monosubstituted probes have bases at and up to a certain number of bases in either direction from the polymorphism, substituted with the remaining nucleotides (selected from A, T, G, C and U). Typically the probes in a tiled detection block will include substitutions of the sequence positions up to and including those that are 5 bases away from the polymorphic site of the biallelic marker. The monosubstituted probes provide internal controls for the tiled array, to distinguish actual hybridization from artefactual cross-hybridization. Upon completion of hybridization with the target sequence and washing of the array, the array is scanned to determine the position on the array to which the target sequence hybridizes. The hybridization data from the scanned array is then analyzed to identify which allele or alleles of the biallelic marker are present in the sample. Hybridization and scanning may be carried out as described in PCT Application No. WO 92/10092 and WO 95/11995 and U.S. Pat. No. 5,424,186, the disclosures of which are incorporated herein by reference in their entireties.

[0321] Thus, in some embodiments, the chips may comprise an array of nucleic acid sequences of fragments of about 15 nucleotides in length. In further embodiments, the chip may comprise an array including at least one of the sequences selected from the group consisting of the nucleic acids of the sequences set forth as SEQ ID Nos 30-75 and the sequences complementary thereto, or a fragment thereof at least about 8 consecutive nucleotides, preferably 10, 15, 20, more preferably 25, 30, or 40 consecutive nucleotides comprising a biallelic marker of the present invention. In some embodiments, the chip may comprise an array of at least 2, 3, 4, 5, 6, 7, 8 or more of these polynucleotides of the invention. Solid supports and polynucleotides of the present invention attached to solid supports are further described in “Oligonucleotide primers and probes”.

[0322] f—Integrated Microsequencing And Capillary Electrophoresis Chips

[0323] Another technique, which may be used to analyze polymorphisms, includes multicomponent integrated systems, which miniaturize and compartmentalize processes such as PCR and capillary electrophoresis reactions in a single functional device. An example of such technique is disclosed in U.S. Pat. No. 5,589,136, the disclosure of which is incorporated herein by reference in its entirety, which describes the integration of PCR amplification and capillary electrophoresis in chips.

[0324] Integrated systems can be envisaged mainly when microfluidic systems are used. These systems comprise a pattern of microchannels designed onto a glass, silicon, quartz, or plastic wafer included on a microchip. The movements of the samples are controlled by electric, electroosmotic or hydrostatic forces applied across different areas of the microchip to create functional microscopic valves and pumps with no moving parts.

[0325] For genotyping biallelic markers, the microfluidic system may integrate nucleic acid amplification, microsequencing, capillary electrophoresis and a detection method such as laser-induced fluorescence detection.

Association Studies with the Biallelic Markers of the RBP-7 Gene

[0326] The identification of genes involved in suspected heterogeneous, polygenic and multifactorial traits such as cancer can be carried out through two main strategies currently used for genetic mapping: linkage analysis and association studies. Association studies examine the frequency of marker alleles in unrelated trait positive (T+) individuals compared with trait negative (T−) controls, and are generally employed in the detection of polygenic inheritance. Association studies as a method of mapping genetic traits rely on the phenomenon of linkage disequilibrium, which is described below.

[0327] If two genetic loci lie on the same chromosome, then sets of alleles of these loci on the same chromosomal segment (called haplotypes) tend to be transmitted as a block from generation to generation. When not broken up by recombination, haplotypes can be tracked not only through pedigrees but also through populations. The resulting phenomenon at the population level is that the occurrence of pairs of specific alleles at different loci on the same chromosome is not random, and the deviation from random is called linkage disequilibrium (LD).

[0328] If a specific allele in a given gene is directly involved in causing a particular trait T, its frequency will be statistically increased in a T+ population when compared to the frequency in a T− population. As a consequence of the existence of LD, the frequency of all other alleles present in the haplotype carrying the trait-causing allele (TCA) will also be increased in T+ individuals compared to T− individuals. Therefore, association between the trait and any allele in linkage disequilibrium with the trait-causing allele will suffice to suggest the presence of a trait-related gene in that particular allele's region. Linkage disequilibrium allows the relative frequencies in T+ and T− populations of a limited number of genetic polymorphisms (specifically biallelic markers) to be analyzed as an alternative to screening all possible functional polymorphisms in order to find trait-causing alleles.

[0329] The general strategy to perform association studies using biallelic markers derived from a candidate region is to scan two groups of individuals (trait+ and trait− control individuals which are characterized by a well defined phenotype as described below) in order to measure and statistically compare the allele frequencies of such biallelic markers in both groups.

[0330] If a statistically significant association with a trait is identified for at least one or more of the analyzed biallelic markers, one can assume that:either the associated allele is directly responsible for causing the trait (associated allele is the TCA), or the associated allele is in LD with the TCA. If the evidence indicates that the associated allele within the candidate region is most probably not the TCA but is in LD with the real TCA, then the TCA, and by consequence the gene carrying the TCA, can be found by sequencing the vicinity of the associated marker.

[0331] It is another object of the present invention to provide a method for the identification and characterization of an association between alleles for one or several biallelic markers of the human RBP-7 gene and a trait. The method comprises the steps of:

[0332] genotyping a marker or a group of biallelic markers according to the invention in trait positive and trait negative individuals; and

[0333] establishing a statistically significant association between one allele of at least one marker and the trait.

[0334] Preferably, the trait positive and trait negative individuals are selected from non-overlapping phenotypes, at opposite ends of the non-bimodal phenotype spectra of the trait under study. In some embodiments, the biallelic marker is one of the biallelic markers of the present invention.

[0335] In a preferred embodiment, the trait is a disease and preferably a cancer.

[0336] The present invention also provides a method for the identification and characterization of an association between a haplotype comprising alleles for several biallelic markers of the human RBP-7 gene and a trait. The method comprises the steps of:

[0337] genotyping a group of biallelic markers according to the invention in trait positive and trait negative individuals; and

[0338] establishing a statistically significant association between a haplotype and the trait.

[0339] In some embodiments, the haplotype comprises two or more biallelic markers defined in SEQ ID Nos 30-71.

[0340] The step of testing for and detecting the presence of DNA comprising specific alleles of a biallelic marker or a group of biallelic markers of the present invention can be carried out as described further below.

Vectors for the Expression of a Regulatory or a Coding Polynucleotide According to the Invention

[0341] Generally, a recombinant vector of the invention may comprise any of the polynucleotides described herein, including regulatory sequences, coding sequences and polynucleotide constructs, as well as any RBP-7 primer or probe as defined above. More particularly, the recombinant vectors of the present invention can comprise any of the polynucleotides described in the “RBP-7 Gene, Corresponding cDNAs And RBP-7 Coding And Regulating Sequences” section, and the “Oligonucleotide Probes And Primers” section.

[0342] Any of the regulatory polynucleotides or the coding polynucleotides of the invention may be inserted into recombinant vectors for expression in a recombinant host cell or a recombinant host organism.

[0343] Thus, the present invention also encompasses a family of recombinant vectors that at contains either a RBP-7 regulatory polynucleotide or a RBP-7 coding polynucleotide or both of them. Preferably, the present invention concerns recombinant vectors that contains either a RBP-7 regulatory polynucleotide or a RBP-7 coding polynucleotide comprising at least one of the biallelic markers of the invention, particularly those of SEQ ID Nos 30-71.

[0344] More particularly, the present invention also relates to expression vectors which include nucleic acids encoding a RBP-7 protein under the control of either a RBP-7 regulatory polynucleotide, or an exogenous regulatory sequence.

[0345] Another aspect of the present invention is a recombinant expression vector comprising a nucleic acid selected from the group consisting of SEQ ID Nos 1, 4, 5-28 or complementary sequences thereto or fragments or variants thereof.

[0346] Another preferred recombinant expression vector according to the invention comprises a nucleic acid comprising a combination of at least two polynucleotides selected from the group consisting of SEQ ID Nos 5-28 or the sequences complementary thereto, wherein the polynucleotides are arranged within the nucleic acid, from the 5′ end to the 3′ end of said nucleic acid, in the same order than in the SEQ ID No. 1.

[0347] Another aspect of the invention is a recombinant expression vector comprising a nucleic acid selected from the group consisting of SEQ ID No. 2 or 3 or the sequences complementary thereto or a biologically active fragment or variant thereof.

[0348] A further aspect of the invention is a recombinant expression vector comprising a purified or isolated nucleic acid comprising:

[0349] a) a nucleic acid comprising the nucleotide sequence SEQ ID No. 2, a fragment or variant thereof or a nucleotide sequence complementary thereto;

[0350] b) a polynucleotide encoding a protein or a polynucleotide of interest.

[0351] The invention also encompasses a recombinant expression vector containing a polynucleotide comprising, consisting essentially of, or consisting of:

[0352] a) a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 2, or the sequence complementary thereto , or a biologically active fragment or variant thereof; and

[0353] b) a polynucleotide encoding a polypeptide or a polynucleotide of interest.

[0354] c) Optionally, the expression vector may further comprise a nucleic acid comprising a regulatory polynucleotide of SEQ ID No. 3, or the sequence complementary thereto, or a biologically active fragment or variant thereof.

[0355] The vector containing the appropriate DNA sequence as described above, more preferably a RBP-7 regulatory polynucleotide, a RBP-7 coding polynucleotide or both of them, can be utilized to transform an appropriate host to allow the expression of the desired polypeptide or polynucleotide.

Vectors

[0356] A recombinant vector according to the invention comprises, but is not limited to, a YAC (Yeast Artificial Chromosome), a BAC (Bacterial Artificial Chromosome), a phage, a phagemid, a cosmid, a plasmid or even a linear DNA molecule which may comprise, consist essentially of, or consist of a chromosomal, non-chromosomal and synthetic DNA. Such a recombinant vector can comprise a transcriptional unit comprising an assembly of

[0357] (1) a genetic element or elements having a regulatory role in gene expression, for example promoters or enhancers. Enhancers are cis-acting elements of DNA, usually from about 10 to 300 bp that act on the promoter to increase the transcription.

[0358] (2) a structural or coding sequence which is transcribed into mRNA and eventually translated into a polypeptide, and

[0359] (3) appropriate transcription initiation and termination sequences. Structural units intended for use in yeast or eukaryotic expression systems preferably include a leader sequence enabling extracellular secretion of translated protein by a host cell. Alternatively, where recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal residue. This residue may or may not be subsequently cleaved from the expressed recombinant protein to provide a final product.

[0360] Generally, recombinant expression vectors will include origins of replication, selectable markers permitting transformation of the host cell, and a promoter derived from a highly expressed gene to direct transcription of a downstream structural sequence. The selectable marker genes can be for example dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, TRP1 for S. cerevisiae or tetracycline, rifampicine or ampicillin resistance in E. coli, or levan saccharase for mycobacteria. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences, and preferably a leader sequence capable of directing secretion of translated protein into the periplasmic space or extracellular medium.

[0361] Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired polypeptide with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.

[0362] As a representative but non-limiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of pBR322 (ATCC 37017). Such commercial vectors include, for example, pKK223-3 (Pharmacia, Uppsala, Sweden), and GEM1 (Promega Biotec, Madison, Wis., USA).

[0363] A suitable vector for the expression of the RBP-7 protein above-defined or their peptide fragments is a baculovirus vector that can be propagated in insect cells and in insect cell lines. A specific suitable host vector system is the pVL1392/1393 baculovirus transfer vector (Pharmingen) that is used to transfect the SF9 cell line (ATCC No. CRL 1711) which is derived from Spodoptera frugiperda. Other baculovirus vectors are described in Chai et al. (1993), Viasak et al. (1983) and Lenhardt et al. (1996).

[0364] Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking nontranscribed sequences. DNA sequences derived from the SV40 viral genome, for example SV40 origin, early promoter, enhancer, splice and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

[0365] Large numbers of suitable vectors and promoters are known to those of skill in the art, and commercially available, such as bacterial vectors: pQE70, pQE60, pQE-9 (Qiagen), pbs, pD10, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16A, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); or eukaryotic vectors: pWLNEO, pSV2CAT, pOG44, pXT1, pSG (Stratagene); pSVK3, pBPV, pMSG, pSVL (Pharmacia); baculovirus transfer vector pVL1392/1393 (Pharmingen); pQE-30 (QIAexpress).

Promoters

[0366] The suitable promoter regions used in the expression vectors according to the present invention are choosen taking into account of the cell host in which the heterologous gene has to be expressed.

[0367] Preferred bacterial promoters are the LacI, LacZ, the T3 or T7 bacteriophage RNA polymerase promoters, the polyhedrin promoter, or the p 10 protein promoter from baculovirus (Kit Novagen) (Smith et al., 1983.; O'Reilly et al., 1992), the lambda P_(R) promoter or also the trc promoter.

[0368] Preferred promoters for the expression of the heterologous gene in eukaryotic hosts are the early promoter of CMV, the Herpes simplex virus thymidine kinase promoter, the early or the late promoter from SV40, the LTR regions of certain retroviruses or also the mouse metallothionein I promoter.

[0369] Promoter regions can be selected from any desired gene using, for example, CAT (chloramphenicol transferase) vectors and more preferably pKK232-8 and pCM7 vectors. Particularly named bacterial promoters include lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-L. Selection of a convenient vector and promoter is well within the level of ordinary skill in the art.

[0370] The choice of a determined promoter, among the above-described promoters is well in the ability of one skill in the art, guided by his knowledge in the genetic engineering technical field, and by being also guided by the book of Sambrook et al. in 1989 or also by the procedures described by Fuller et al. in 1996.

Other Types of Vectors

[0371] The in vivo expression of a RBP-7 polypeptide or a fragment or a variant thereof may be useful in order to study the physiological consequences of a deregulation of its in vivo synthesis on the physiology of the recipient recombinant host organism under study, more particularly on the cell differentiation and on an eventual abnormal proliferation of various kinds of cells, including T cells and epithelial cells.

[0372] Consequently, the present invention also relates to recombinant expression vectors mainly designed for the in vivo production of a therapeutic peptide fragment by the introduction of the genetic information in the organism of the patient to be treated. This genetic information may be introduced in vitro in a cell that has been previously extracted from the organism, the modified cell being subsequently reintroduced in the said organism, directly in vivo into the appropriate tissue.

[0373] The method for delivering the corresponding protein or peptide to the interior of a cell of a vertebrate in vivo comprises the step of introducing a preparation comprising a physiologically acceptable carrier and a naked polynucleotide operatively coding for the polypeptide into the interstitial space of a tissue comprising the cell, whereby the naked polynucleotide is taken up into the interior of the cell and has a physiological effect.

[0374] In a specific embodiment, the invention provides a composition for the in vivo production of a RBP-7 polypeptide containing a naked polynucleotide operatively coding for a RBP-7 polypeptide or a fragment or a variant thereof, in solution in a physiologically acceptable carrier and suitable for introduction into a tissue to cause cells of the tissue to express the said protein or polypeptide.

[0375] Advantageously, the composition described above is administered locally, near the site in which the expression of a RBP-7 polypeptide or a fragment or a variant thereof is sought.

[0376] The polynucleotide operatively coding for a RBP-7 polypeptide or a fragment or variant thereof may be a vector comprising the genomic DNA or the complementary DNA (cDNA) coding for the corresponding protein or its protein derivative and a promoter sequence allowing the expression of the genomic DNA or the complementary DNA in the desired eukaryotic cells, such as vertebrate cells, specifically mammalian cells.

[0377] The promoter contained in such a vector is selected among the group comprising:

[0378] an internal or an endogenous promoter, such as the natural promoter associated with the structural gene coding for the desired RBP-7 polypeptide or the fragment or variant thereof; such a promoter may be completed by a regulatory element derived from the vertebrate host, in particular an activator element;

[0379] a promoter derived from a cytoskeletal protein gene such as the desmin promoter (Bolmont et al., 1990; Zhenlin et al., 1989).

[0380] As a general feature, the promoter may be heterologous to the vertebrate host, but it is advantageously homologous to the vertebrate host.

[0381] By a promoter heterologous to the vertebrate host is intended a promoter that is not found naturally in the vertebrate host.

[0382] Compositions comprising a polynucleotide are described in the PCT Application No. WO 90/11092 and also in the PCT Application No. WO 95/11307 as well as in the articles of Tacson et al. (1996) and of Huygen et al. (1996), the disclosures of which are incorporated herein by reference in their entireties.

[0383] In another embodiment, the DNA to be introduced is complexed with DEAE-dextran (Pagano et al., 1967) or with nuclear proteins (Kaneda et al., 1989), with lipids (Felgner et al., 1987) or encapsulated within liposomes (Fraley et al., 1980).

[0384] In another embodiment, the polynucleotide encoding a RBP-7 polypeptide or a fragment or a variant thereof may be included in a transfection system comprising polypeptides that promote its penetration within the host cells as it is described in the PCT Application WO 95/10534, the disclosure of which is incorporated herein by reference in its entirety.

[0385] The vector according to the present invention may advantageously be administered in the form of a gel that facilitates their transfection into the cells. Such a gel composition may be a complex of poly-L-lysine and lactose, as described by Midoux (1993) or also poloxamer 407 as described by Pastore (1994). Said vector may also be suspended in a buffer solution or be associated with liposomes.

[0386] The amount of the vector to be injected to the desired host organism vary according to the site of injection. As an indicative dose, it will be injected between 0,1 and 100 μg of the vector in an animal body, preferably a mammal body, for example a mouse body.

[0387] In another embodiment of the vector according to the invention, said vector may be introduced in vitro in a host cell, preferably in a host cell previously harvested from the animal to be treated and more preferably a somatic cell such as a muscle cell. In a subsequent step, the cell that has been transformed with the vector coding for the desired RBP-7 polypeptide or the desired fragment or variant thereof is implanted back into the animal body in order to deliver the recombinant protein within the body either locally or systemically.

[0388] Suitable vectors for the in vivo expression of a RBP-7 polypeptide or a fragment or a variant thereof are described hereunder.

[0389] In one specific embodiment, the vector is derived from an adenovirus. Preferred adenovirus vectors according to the invention are those described by Feldman and Steg (1996) or Ohno et al. (1994). Another preferred recombinant adenovirus according to this specific embodiment of the present invention is the adenovirus described by Ohwada et al. (1996) or the human adenovirus type 2 or 5 (Ad 2 or Ad 5) or an adenovirus of animal origin ( French Patent Application No. FR-93.05954, the disclosure of which is incorporated herein by reference in its entirety).

[0390] Among the adenoviruses of animal origin it can be cited the adenoviruses of canine (CAV2, strain Manhattan or A26/61[ATCC VR-800]), bovine, murine (Mavl, Beard et al., 1980) or simian (SAV). Other adenoviruses are described by Levrero et al. (1991), Graham et al. (1984), in the European Patent Application No. EP-185.573 or in the PCT Application No. WO 95/14785, the disclosures of which are incorporated herein by reference in their entireties.

[0391] Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous polynucleotides in vivo , particularly to mammals, including humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host. Suitable retroviruses used according to the present invention include those described in the PCT Application No. WO 93/25234, the PCT Application No. WO 94/06920, the PCT Application No. WO 94/ 24298, Roth et al. (1996), Roux et al. (1989), Julian et al. (1992) and Neda et al. (1991), the disclosures of which are incorporated herein by reference in their entireties. Other preferred retrovirus include Murine Leukemia Viruses such as 4070A and 1504A (Hartley et al., 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Gross (ATCC No. VR-590), Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190; PCT Application No. WO 94/24298), the disclosure of which is incorporated herein by reference in its entirety, and also Rous Sarcoma Viruses such as Bryan high titer (ATCC Nos VR-334, VR-657, VR-726, VR-659 and VR-728.

[0392] Yet another viral vector system that is contemplated by the invention comprises the adeno-associated virus (AAV). Adeno-associated virus is a naturally occuring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle (Muzyczka et al., 1992). It is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (Flotte et al., 1992; Samulski et al., 1989; McLaughlin et al., 1989). One advantageous feature of AAV derives from its reduced efficacy for transducing primary cells relative to transformed cells.

[0393] Other compositions containing a vector of the invention comprise advantageously an oligonucleotide fragment of the nucleic sequence of RBP-7 as an antisense tool that inhibits the expression of the corresponding gene and is thus useful to inhibit the expression of the RBP-7 gene in the tagged cells or organs. Preferred methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al. (1995) or also in the PCT Application No. WO 95/24223, the disclosure of which is incorporated herein by reference in its entirety.

Vectors Suitable for Homologous Recombination

[0394] Other suitable vectors, particularly for the expression of genes in mammalian cells, may be selected from the group of vectors consisting of P1 bacteriophages, and bacterial artificial chromosomes (BACs). These types of vectors may contain large inserts ranging from about 80-90 kb (P1 bacteriophage) to about 300 kb (BACs).

P1 bacteriophage.

[0395] The construction of P1 bacteriophage vectors such as p158 or p158/neo8 are notably described by Sternberg (1992, 1994). Recombinant P1 clones comprising RBP-7 nucleotide sequences may be designed for inserting large polynucleotides of more than 40 kb (Linton et al., 1993). To generate P1 DNA for transgenic experiments, a preferred protocol is the protocol described by McCormick et al. (1994). Briefly, E. coli (preferably strain NS3529) harboring the P1 plasmid are grown overnight in a suitable broth medium containing 25 μg/ml of kanamycin. The P1 DNA is prepared from the E. coli by alkaline lysis using the Qiagen Plasmid Maxi kit (Qiagen, Chatsworth, Calif., USA), according to the manufacturer's instructions. The P1 DNA is purified from the bacterial lysate on two Qiagen-tip 500 columns, using the washing and elution buffers contained in the kit. A phenol/chloroform extraction is then performed before precipitating the DNA with 70% ethanol. After solubilizing the DNA in TE (10 mM Tris-HCl, pH 7.4, 1 mM EDTA), the concentration of the DNA is assessed by spectrophotometry.

[0396] When the goal is to express a P1 clone comprising RBP-7 nucleotide sequences in a transgenic animal, typically in transgenic mice, it is desirable to remove vector sequences from the P1 DNA fragment, for example by cleaving the P1 DNA at rare-cutting sites within the P1 polylinker (SfiI, NotI or SalI). The P1 insert is then purified from vector sequences on a pulsed-field agarose gel, using methods similar using methods similar to those originally reported for the isolation of DNA from YACs (Schedl et al., 1993a; Peterson et al., 1993). At this stage, the resulting purified insert DNA can be concentrated, if necessary, on a Millipore Ultrafree-MC Filter Unit (Millipore, Bedford, Mass., USA 30,000 molecular weight limit) and then dialyzed against microinjection buffer (10 mM Tris-HCl, pH 7.4; 250 μM EDTA) containing 100 mM NaCl, 30 μM spermine, 70 μM spermidine on a microdyalisis membrane (type VS, 0.025 μM from Millipore). The intactness of the purified P1 DNA insert is assessed by electrophoresis on 1% agarose (Sea Kem GTG; FMC Bio-products) pulse-field gel and staining with ethidium bromide.

Bacterial Artificial Chromosomes (BACs)

[0397] The bacterial artificial chromosome (BAC) cloning system (Shizuya et al., 1992) has been developed to stably maintain large fragments of genomic DNA (100-300 kb) in E. coli. A preferred BAC vector is the pBeloBAC11 vector that has been described by Kim et al. (1996) BAC libraries are prepared with this vector using size-selected genomic DNA that has been partially digested using enzymes that permit ligation into either the Bam HI or HindIII sites in the vector. Flanking these cloning sites are T7 and SP6 RNA polymerase transcription initiation sites that can be used to generate end probes by either RNA transcription or PCR methods. After the construction of a BAC library in E. coli, BAC DNA is purified from the host cell as a supercoiled circle. Converting these circular molecules into a linear form precedes both size determination and introduction of the BACs into recipient cells. The cloning site is flanked by two Not I sites, permitting cloned segments to be excised from the vector by Not I digestion. Alternatively, the DNA insert contained in the pBeloBAC 11 vector may be linearized by treatment of the BAC vector with the commercially available enzyme lambda terminase that leads to the cleavage at the unique cosN site, but this cleavage method results in a full length BAC clone containing both the insert DNA and the BAC sequences.

Specific DNA Construct Vector for Homologous Recombination

[0398] The term “DNA construct” is understood to mean a linear or circular purified or isolated polynucleotide that has been artificially designed and which comprises at least two nucleotide sequences that are not found as contiguous nucleotide sequences in their natural environment.

DNA Construct that Enables Directing Temporal and Spatial Gene Expression in Recombinant Cell Hosts and in Transgenic Animals

[0399] In order to study the physiological and phenotype consequences of a lack of synthesis of the RBP-7 protein, both at the cell level and at the multi cellular organism level, in particular as regards to disorders related to abnormal cell proliferation, notably cancers, the invention also encompasses DNA constructs and recombinant vectors enabling a conditional expression of a specific allele of the RBP-7 genomic sequence or cDNA and also of a copy of this genomic sequence or cDNA harboring substitutions, deletions, or additions of one or more bases as regards to the RBP-7 nucleotide sequence of SEQ ID Nos 1 or 4, or a fragment thereof, these base substitutions, deletions or additions being located either in an exon, an intron or a regulatory sequence, but preferably in the 5′-regulatory sequence or in an exon of the RBP-7 genomic sequence or within the RBP-7 cDNA of SEQ ID No. 4.

[0400] A first preferred DNA construct is based on the tetracycline resistance operon tet from E. coli transposon Tn110 for controlling the RBP-7 gene expression, such as described by Gossen et al. (1992, 1995) and Furth et al. (1994). Such a DNA construct contains seven tet operator sequences from Tn10 (tetop) that are fused to either a minimal promoter or a 5′-regulatory sequence of the RBP-7 gene, said minimal promoter or said RBP-7 regulatory sequence being operably linked to a polynucleotide of interest that codes either for a sense or an antisense oligonucleotide or for a polypeptide, including a RBP-7 polypeptide or a peptide fragment thereof. This DNA construct is functional as a conditional expression system for the nucleotide sequence of interest when the same cell also comprises a nucleotide sequence coding for either the wild type (tTA) or the mutant (rTA) repressor fused to the activating domain of viral protein VP16 of herpes simplex virus, placed under the control of a promoter, such as the HCMVIE1 enhancer/promoter or the MMTV-LTR. Indeed, a preferred DNA construct of the invention will comprise both the polynucleotide containing the tet operator sequences and the polynucleotide containing a sequence coding for the tTA or the rTA repressor.

[0401] In the specific embodiment wherein the conditional expression DNA construct contains the sequence encoding the mutant tetracycline repressor rTA, the expression of the polynucleotide of interest is silent in the absence of tetracycline and induced in its presence. DNA Constructs Allowing Homologous Recombination:Replacement Vectors

[0402] A second preferred DNA construct will comprise, from 5′-end to 3′-end: (a) a first nucleotide sequence that is comprised in the RBP-7 genomic sequence; (b) a nucleotide sequence comprising a positive selection marker, such as the marker for neomycine resistance (neo); and (c) a second nucleotide sequence that is comprised in the RBP-7 genomic sequence, and is located on the genome downstream the first RBP-7 nucleotide sequence (a).

[0403] In a preferred embodiment, this DNA construct also comprises a negative selection marker located upstream the nucleotide sequence (a) or downstream the nucleotide sequence (b). Preferably, the negative selection marker is the thymidine kinase (tk) gene (Thomas et al., 1986), the hygromycine beta gene (Te Riele et al., 1990), the hprt gene ( Van der Lugt et al., 1991; Reid et al., 1990) or the Diphteria toxin A fragment (Dt-A) gene (Nada et al., 1993; Yagi et al. 1990). Preferably, the positive selection marker is located within a RBP-7 exon sequence so as to interrupt the sequence encoding a RBP-7 protein.

[0404] These replacement vectors are described for example by Thomas et al. (1986; 1987), Mansour et al. (1988) and Koller et al. (1992).

[0405] The first and second nucleotide sequences (a) and (c) may be indifferently located within a RBP-7 regulatory sequence, an intronic sequence, an exon sequence or a sequence containing both regulatory and/or intronic and/or exon sequences. The size of the nucleotide sequences (a) and (c) is ranging from 1 to 50 kb, preferably from 1 to 10 kb, more preferably from 2 to 6 kb and most preferably from 2 to 4 kb.

DNA Constructs Allowing Homologous Recombination: Cre-Loxp System

[0406] These new DNA constructs make use of the site specific recombination system of the P1 phage. The P1 phage possesses a recombinase called Cre which interacts specifically with a 34 base pairs loxP site. The loxP site is composed of two palindromic sequences of 13 bp separated by a 8 bp conserved sequence (Hoess et al., 1986). The recombination by the Cre enzyme between two loxP sites having an identical orientation leads to the deletion of the DNA fragment.

[0407] The Cre-loxP system used in combination with a homologous recombination technique has been first described by Gu et al. (1993, 1994). Briefly, a nucleotide sequence of interest to be inserted in a targeted location of the genome harbors at least two loxP sites in the same orientation and located at the respective ends of a nucleotide sequence to be excised from the recombinant genome. The excision event requires the presence of the recombinase (Cre) enzyme within the nucleus of the recombinant cell host. The recombinase enzyme may be brought at the desired time either by (a) incubating the recombinant cell hosts in a culture medium containing this enzyme, by injecting the Cre enzyme directly into the desired cell, such as described by Araki et al. (1995), or by lipofection of the enzyme into the cells, such as described by Baubonis et al. (1993); (b) transfecting the cell host with a vector comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter being optionally inducible, said vector being introduced in the recombinant cell host, such as described by Gu et al. (1993) and Sauer et al. (1988); (c) introducing in the genome of the cell host a polynucleotide comprising the Cre coding sequence operably linked to a promoter functional in the recombinant cell host, which promoter is optionally inducible, and said polynucleotide being inserted in the genome of the cell host either by a random insertion event or an homologous recombination event, such as described by Gu et al. (1994).

[0408] In the specific embodiment wherein the vector containing the sequence to be inserted in the RBP-7 gene by homologous recombination is constructed in such a way that selectable markers are flanked by loxP sites of the same orientation, it is possible, by treatment by the Cre enzyme, to eliminate the selectable markers while leaving the RBP-7 sequences of interest that have been inserted by an homologous recombination event. Again, two selectable markers are needed : a positive selection marker to select for the recombination event and a negative selection marker to select for the homologous recombination event. Vectors and methods using the Cre-loxP system are described by Zou et al. (1994).

[0409] Thus, a third preferred DNA construct of the invention comprises, from 5′-end to 3′-end : (a) a first nucleotide sequence that is comprised in the RBP-7 genomic sequence; (b) a nucleotide sequence comprising a polynucleotide encoding a positive selection marker, said nucleotide sequence comprising additionally two sequences defining a site recognized by a recombinase, such as a loxP site, the two sites being placed in the same orientation; and (c) a second nucleotide sequence that is comprised in the RBP-7 genomic sequence, and is located on the genome downstream of the first RBP-7 nucleotide sequence (a).

[0410] The sequences defining a site recognized by a recombinase, such as a loxP site, are preferably located within the nucleotide sequence (b) at suitable locations bordering the nucleotide sequence for which the conditional excision is sought. In one specific embodiment, two loxP sites are located at each side of the positive selection marker sequence, in order to allow its excision at a desired time after the occurrence of the homologous recombination event.

[0411] In a preferred embodiment of a method using the third DNA construct described above, the excision of the polynucleotide fragment bordered by the two sites recognized by a recombinase, preferably two loxP sites, is performed at a desired time, due to the presence within the genome of the recombinant cell host of a sequence encoding the Cre enzyme operably linked to a promoter sequence, preferably an inducible promoter, more preferably a tissue-specific promoter sequence and most preferably a promoter sequence which is both inducible and tissue-specific, such as described by Gu et al. (1994).

[0412] The presence of the Cre enzyme within the genome of the recombinant cell host may result of the breeding of two transgenic animals, the first transgenic animal bearing the RBP-7-derived sequence of interest containing the loxP sites as described above and the second transgenic animal bearing the Cre coding sequence operably linked to a suitable promoter sequence, such as described by Gu et al. (1994).

[0413] Spatio-temporal control of the Cre enzyme expression may also be achieved with an adenovirus based vector that contains the Cre gene thus allowing infection of cells, or in vivo infection of organs, for delivery of the Cre enzyme, such as described by Anton and Graham (1995) and Kanegae et al. (1995).

[0414] The DNA constructs described above may be used to introduce a desired nucleotide sequence of the invention, preferably a RBP-7 genomic sequence or a RBP-7 cDNA sequence, and most preferably an altered copy of a RBP-7 genomic or cDNA sequence, within a predetermined location of the targeted genome, leading either to the generation of an altered copy of a targeted gene (knock-out homologous recombination) or to the replacement of a copy of the targeted gene by another copy sufficiently homologous to allow an homologous recombination event to occur (knock-in homologous recombination).

Nuclear Antisense DNA Constructs

[0415] Preferably, the antisense polynucleotides of the invention have a 3′ polyadenylation signal that has been replaced with a self-cleaving ribozyme sequence, such that RNA polymerase II transcripts are produced without poly(A) at their 3′ ends, these antisense polynucleotides being incapable of export from the nucleus, such as described by Liu et al. (1994). In a preferred embodiment, these RBP-7 antisense polynucleotides also comprise, within the ribozyme cassette, a histone stem-loop structure to stabilize cleaved transcripts against 3′-5′ exonucleolytic degradation, such as described by Eckner et al. (1991).

Cell Hosts

[0416] Another aspect of the invention is a host cell that has been transformed or transfected with one of the polynucleotides described herein, and in particular a polynucleotide either comprising a RBP-7 regulatory polynucleotide or the coding sequence of the RBP-7 polypeptide selected from the group consisting of SEQ ID Nos 1 and 4 or a fragment or a variant thereof. Also included are host cells that are transformed (prokaryotic cells) or that are transfected (eukaryotic cells) with a recombinant vector such as one of those described above. More particularly, the cell hosts of the present invention can comprise any of the polynucleotides described in the “RBP-7 Gene, Corresponding cDNAs And RBP-7 Coding And Regulating Sequences” section, and the “Oligonucleotide Probes And Primers” section.

[0417] A further recombinant cell host according to the invention comprises a polynucleotide containing a biallelic marker selected from the group consisting of A1 to A21, and the complements thereof.

[0418] An additional recombinant cell host according to the invention comprises any of the vectors described herein, more particularly any of the vectors described in the “Vectors For The Expression Of A Regulatory Or A Coding Polynucleotide According To The Invention” section.

[0419] All the above-described vectors are useful to transform or transfect cell hosts in order to express a polynucleotide coding for a RBP-7 polypeptide or their peptide fragments or variants, or a polynucleotide of interest derived from the RBP-7 gene.

[0420] Suitable prokaryotic hosts for transformation include E. coli , Bacillus subtilis, as well as various species within the genera of Streptomyces or Mycobacterium. Suitable eukaryotic hosts comprise yeast, insect cells, such as Drosophila and Sf9. Various mammalian cell hosts can also be employed to express recombinant protein. Examples of mammalian cell hosts include the COS-7 lines of monkey kidney fibroblasts (Guzman, 1981), and other cell lines capable of expressing a compatible vector, for example the C127, 3T3, CHO, HeLa and BHK cell lines. The selection of an host is within the scope of the one skilled in the art.

[0421] A cell host according to the present invention is characterized in that its genome or genetic background (including chromosome, plasmids) is modified by the heterologous nucleic acid coding for a RBP-7 polypeptide or a peptide fragment or variant, or by a polynucleotide of interest derived from the RBP-7 gene.

[0422] Preferred cell hosts used as recipients for the expression vectors of the invention are the followings:

[0423] a) Prokaryotic cells: Escherichia coli strains (I.E. DH5-□ strain) or Bacillus subtilis.

[0424] b) Eukaryotic cell hosts: HeLa cells (ATCC No. CCL2; No. CCL2.1; No. CCL2.2), Cv 1 cells (ATCC No. CCL70), COS cells (ATCC No. CRL1650; No. CRL1651), Sf-9 cells (ATCC No. CRL1711), mammal ES stem cells.

[0425] Preferably, the mammal ES stem cells include human (Thomson et al., 1998), mice, rats and rabbits ES stem cells and are preferably used in a process for producing transgenic animals, such as those described below.

[0426] The RBP-7 gene expression in human cells may be rendered defective, or alternatively it may be proceeded with the insertion of a RBP-7 genomic or cDNA sequence with the replacement of the RBP-7 gene counterpart in the genome of an animal cell by a RBP-7 polynucleotide according to the invention. These genetic alterations may be generated by homologous recombination events using specific DNA constructs that have been previously described.

[0427] One kind of cell hosts that may be used are mammal zygotes, such as murine zygotes. For example, murine zygotes may undergo microinjection with a purified DNA molecule of interest, for example a purified DNA molecule that has previously been adjusted to a concentration range from 1 ng/ml—for BAC inserts-3 ng/μl—for P1 bacteriophage inserts—in 10 mM Tris-HCl, pH 7.4, 250 μM EDTA containing 100 mM NaCl, 30 μM spermine, and 70 μM spermidine. When the DNA to be microinjected has a large size, polyamines and high salt concentrations can be used in order to avoid mechanical breakage of this DNA, as described by Schedl et al (1993b).

[0428] Anyone of the polynucleotides of the invention, including the DNA constructs described herein, may be introduced in an embryonic stem (ES) cell line, preferably a mouse ES cell line. ES cell lines are derived from pluripotent, uncommited cells of the inner cell mass of pre-implantation blastocysts. Peferred ES cell lines are the following: ES-E14TG2a (ATCC No. CRL-1821), ES-D3 (ATCC No. CRL1934 and No. CRL-11632), YS001 (ATCC No. CRL-11776), 36.5 (ATCC No. CRL-11116). To maintain ES cells in an uncommitted state, they are cultured in the presence of growth inhibited feeder cells which provide the appropriate signals to preserve this embryonic phenotype and serve as a matrix for ES cell adherence. Preferred feeder cells are primary embryonic fibroblasts that are established from tissue of day 13- day 14 embryos of virtually any mouse strain, that are maintained in culture, such as described by Abbondanzo et al. (1993) and are inhibited in growth by irradiation, such as described by Robertson (1987), or by the presence of an inhibitory concentration of LIF, such as described by Pease and Williams (1990).

[0429] The constructs in the host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.

[0430] Following transformation of a suitable host and growth of the host to an appropriate cell density, the selected promoter is induced by appropriate means, such as temperature shift or chemical induction, and cells are cultivated for an additional period.

[0431] Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

[0432] Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known by the skill artisan.

Transgenic Animals

[0433] The terms “transgenic animals” or “host animals” are used herein designate animals that have their genome genetically and artificially manipulated so as to include one of the nucleic acids according to the invention. Preferred animals are non-human mammals and include those belonging to a genus selected from Mus (e.g. mice), Rattus (e.g. rats) and Oryctogalus (e.g. rabbits) which have their genome artificially and genetically altered by the insertion of a nucleic acid according to the invention.

[0434] The transgenic animals of the invention all include within a plurality of their cells a cloned recombinant or synthetic DNA sequence, more specifically one of the purified or isolated nucleic acids comprising a RBP-7 coding sequence, a RBP-7 regulatory polynucleotide or a DNA sequence encoding an antisense polynucleotide such as described in the present specification.

[0435] Preferred transgenic animals according to the invention contains in their somatic cells and/or in their germ line cells any one of the polynucleotides, the recombinant vectors and the cell hosts described in the present invention. More particularly, the transgenic animals of the present invention can comprise any of the polynucleotides described in the “RBP-7 Gene, Corresponding cDNAs And RBP-7 Coding And Regulating Sequences” section, the “Oligonucleotide Probes And Primers” section, the “Vectors For The Expression Of A Regulatory Or A Coding Polynucleotide According To The Invention” section and the “Cell Hosts” section.

[0436] The transgenic animals of the invention thus contain specific sequences of exogenous genetic material such as the nucleotide sequences described above in detail.

[0437] In a first preferred embodiment, these transgenic animals may be good experimental models in order to study the diverse pathologies related to cell differentiation, in particular concerning the transgenic animals within the genome of which has been inserted one or several copies of a polynucleotide encoding a native RBP-7 protein, or alternatively a mutant RBP-7 protein.

[0438] In a second preferred embodiment, these transgenic animals may express a desired polypeptide of interest under the control of the regulatory polynucleotides of the RBP-7 gene, leading to good yields in the synthesis of this protein of interest, and eventually a tissue specific expression of this protein of interest.

[0439] The design of the transgenic animals of the invention may be made according to the conventional techniques well known from the one skilled in the art. For more details regarding the production of transgenic animals, and specifically transgenic mice, it may be referred to Sandou et al. (1994) and also to U.S. Pat. Nos. 4,873,191, issued Oct. 10, 1989, 5,464,764 issued Nov. 7, 1995 and 5,789,215, issued Aug. 4, 1998, these documents being herein incorporated by reference to disclose methods producing transgenic mice.

[0440] Transgenic animals of the present invention are produced by the application of procedures which result in an animal with a genome that has incorporated exogenous genetic material. The procedure involves obtaining the genetic material, or a portion thereof, which encodes either a RBP-7 coding sequence, a RBP-7 regulatory polynucleotide or a DNA sequence encoding a RBP-7 antisense polynucleotide such as described in the present specification.

[0441] A recombinant polynucleotide of the invention is inserted into an embryonic or ES stem cell line. The insertion is preferably made using electroporation, such as described by Thomas et al. (1987). The cells subjected to electroporation are screened (e.g. by selection via selectable markers, by PCR or by Southern blot analysis) to find positive cells which have integrated the exogenous recombinant polynucleotide into their genome, preferably via an homologous recombination event. An illustrative positive-negative selection procedure that may be used according to the invention is described by Mansour et al. (1988).

[0442] Then, the positive cells are isolated, cloned and injected into 3.5 days old blastocysts from mice, such as described by Bradley (1987). The blastocysts are then inserted into a female host animal and allowed to grow to term.

[0443] Alternatively, the positive ES cells are brought into contact with embryos at the 2.5 days old 8-16 cell stage (morulae) such as described by Wood et al. (1993) or by Nagy et al. (1993), the ES cells being internalized to colonize extensively the blastocyst including the cells which will give rise to the germ line.

[0444] The offsprings of the female host are tested to determine which animals are transgenic e.g. include the inserted exogenous DNA sequence and which are wild-type.

[0445] Thus, the present invention also concerns a transgenic animal containing a nucleic acid, a recombinant expression vector or a recombinant host cell according to the invention.

Recombinant Cell Lines Derived from the Transgenic Animals of the Invention

[0446] A further aspect of the invention is recombinant cell hosts obtained from a transgenic animal described herein.

[0447] Recombinant cell lines may be established in vitro from cells obtained from any tissue of a transgenic animal according to the invention, for example by transfection of primary cell cultures with vectors expressing onc-genes such as SV40 large T antigen, as described by Chou (1989) and Shay et al. (1991).

RBP-7 Polypeptides

[0448] It is now easy to produce proteins in high amounts by genetic engineering techniques through expression vectors such as plasmids, phages or phagemids. The polynucleotide that code for one the polypeptides of the present invention is inserted in an appropriate expression vector in order to produce in vitro the polypeptide of interest.

[0449] Thus, the present invention also concerns a method for producing one of the polypeptides described herein, and especially a polypeptide of SEQ ID No. 29 or a fragment or a variant thereof, wherein said method comprises the steps of:

[0450] a) Optionally amplifying the nucleic acid coding for a RBP-7 polypeptide, or a fragment or a variant thereof, using a pair of primers according to the invention (by PCR, SDA, TAS, 3SR NASBA, TMA etc.).

[0451] b) Inserting the resulting amplified nucleic acid in an appropriate vector;

[0452] c) culturing, in an appropriate culture medium, a cell host previously transformed or transfected with the recombinant vector of step b);

[0453] d) harvesting the culture medium thus conditioned or lyse the cell host, for example by sonication or by an osmotic shock;

[0454] e) separating or purifying, from the said culture medium, or from the pellet of the resultant host cell lysate the thus produced polypeptide of interest.

[0455] f) Optionally characterizing the produced polypeptide of interest.

[0456] The polypeptides according to the invention may be characterized by binding onto an immunoaffinity chromatography column on which polyclonal or monoclonal antibodies directed to a polypeptide of SEQ ID No. 29, or a fragment or a variant thereof, have previously been immobilized.

[0457] Purification of the recombinant proteins or peptides according to the present invention may be carried out by passage onto a Nickel or Cupper affinity chromatography column. The Nickel chromatography column may contain the Ni-NTA resin (Porath et al., 1975).

[0458] The polypeptides or peptides thus obtained may be purified, for example by high performance liquid chromatography, such as reverse phase and/or cationic exchange HPLC, as described by Rougeot et al. (1994). The reason to prefer this kind of peptide or protein purification is the lack of byproducts found in the elution samples which renders the resultant purified protein or peptide more suitable for a therapeutic use.

[0459] Another aspect of the present invention comprises a purified or isolated RBP-7 polypeptide or a fragment or a variant thereof.

[0460] In a preferred embodiment, the RBP-7 polypeptide comprises an amino acid sequence of SEQ ID No. 29 or a fragment or a variant thereof. In a further embodiment, the present invention embodies isolated, purified, and recombinant polypeptides comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 29.

[0461] The RBP-7 polypeptide of the amino acid sequence of SEQ ID No. 29 has 1312 amino acids in length. This 1312 amino acid sequence harbors notably potential sites indicating post-translational modifications such as 8 N-glycosylation sites, 72 phosphorylation sites, 8 N-myristoylation sites and 4 amidation sites. The location of these sites is referred to in the appended Sequence Listing when disclosing the features of the amino acid sequence of SEQ ID No. 29.

[0462] The RBP-7 polypeptide shares some homology in amino acid sequence with another retinoblastoma binding protein, namely human RBP-1 (Fattaey et al., 1993). More precisely, a 48% identity has been found between RBP-7 and RBP-1 for the amino acid sequence beginning at position 1 and ending at position 790 of RBP-7. A 30% identity has been found for the amino acid sequence beginning at position 791 and ending at position 1312 of RBP-7.

[0463] A further object of the present invention concerns a purified or isolated polypeptide which is encoded by a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID Nos 1, 4 and 5-28 or fragments or variants thereof. Preferably, the purified or isolated polypeptide comprises at least 10, at least 15, at least 20 or at least 25 consecutive amino acids of the polypeptides encoded by SEQ ID Nos 1, 4 and 5-28.

[0464] The invention includes a nucleic acid encoding a RBP-7 polypeptide comprising at least one of the biallelic markers of the present invention, more particularly at least one of the biallelic markers defined in SEQ ID No. 30-71.

[0465] More generally, the invention also pertains to a variant RBP-7 polypeptide comprising at least one amino acid substitution, addition or deletion, when compared with the sequence of SEQ ID No. 29. More particularly, the invention encompasses a RBP-7 protein or a fragment thereof comprising a contiguous span of at least 6 amino acids, preferably at least 8 to 10 amino acids, more preferably at least 12, 15, 20, 25, 30, 40, 50, or 100 amino acids of SEQ ID No. 29 comprising at least one of the following amino acids:

[0466] a Glycine residue at the amino acid position 293 of SEQ ID No. 29;

[0467] a Glutamic acid at the amino acid in position 963 of SEQ ID No. 29;

[0468] a Methionine residue at the amino acid position 969 of SEQ ID No. 29.

[0469] A variant or mutated RBP-7 polypeptide comprises amino acid changes of at least one amino acid substitution, deletion or addition, preferably from 1 to 10, 20 or 30 amino acid substitutions or additions. The amino acid substitutions are generally non conservative in terms of polarity, charge, hydrophilicity properties of the substitute amino acid when compared with the native amino acid. The amino acid changes occurring in such a mutated RBP-7 polypeptide may be determinant for the biological activity or for the capacity of the mutated RBP-7 polyeptide to be recognized by antibodies raised against a native RBP-7.

[0470] Such a variant or mutated RBP-7 protein may be the target of diagnostic tools, such as specific monoclonal or polyclonal antibodies, useful for detecting the mutated RBP-7 protein in a sample.

[0471] Are also part of the present invention polypeptides that are homologous to a RBP-7 polypeptide, especially a polypeptide of SEQ ID No. 29, or their fragments or variants.

[0472] The invention also encompasses a RBP-7 polypeptide or a fragment or a variant thereof in which at least one peptide bound has been modified as described in “Definitions”.

[0473] The polypeptides according to the invention may also be prepared by the conventional methods of chemical synthesis, either in a homogenous solution or in solid phase. As an illustrative embodiment of such chemical polypeptide synthesis techniques, it may be cited the homogenous solution technique described by Houbenweyl in 1974.

[0474] The RBP-7 polypeptide, or a fragment or a variant thereof may thus be prepared by chemical synthesis in liquid or solid phase by successive couplings of the different amino acid residues to be incorporated (from the N-terminal end to the C-terminal end in liquid phase, or from the C-terminal end to the N-terminal end in solid phase) wherein the N-terminal ends and the reactive side chains are previously blocked by conventional groups.

[0475] For solid phase synthesis the technique described by Merrifield (1965) may be used in particular.

Antibodies

[0476] The polypeptides according to the present invention, especially the polypeptides of SEQ ID No. 29 are allowing the preparation of polyclonal or monoclonal antibodies that recognize the polypeptides of SEQ ID No. 29 or fragments thereof.

[0477] The antibodies may be prepared from hybridomas according to the technique described by Kohler and Milstein in 1975. The polyclonal antibodies may be prepared by immunization of a mammal, especially a mouse or a rabbit, with a polypeptide according to the invention that is combined with an adjuvant of immunity, and then by purifying of the specific antibodies contained in the serum of the immunized animal on a affinity chromatography column on which has previously been immobilized the polypeptide that has been used as the antigen.

[0478] The invention also concerns a purified or isolated antibody capable of specifically binding to the RBP-7 protein, more particularly to selected peptide fragments thereof, and more preferably polypeptides encoded by nucleic acids comprising one or more biallelic markers of the invention, or a variant thereof. In addition, the invention comprises antibodies capable of specifically binding to a fragment or variant of such a RBP-7 protein comprising an epitope of the RBP-7 protein, preferably an antibody capable of binding to a polypeptide comprising at least 10 consecutive amino acids, at least 15 consecutive amino acids, at least 20 consecutive amino acids, or at least 40 consecutive amino acids of a RBP-7 protein, more preferably an antibody capable of binding specifically to a variant or mutated RBP-7 protein or a fragment thereof and distinguishing between either two variants of RBP-7 or mutated RBP-7 and non-mutated RBP-7 protein.

[0479] The proteins expressed from a RBP-7 DNA comprising at least one of the nucleic sequences of SEQ ID Nos 30-71 or a fragment or a variant thereof, preferably the nucleic sequences of the biallelic markers leading to an amino acid substitution, may also be used to generate antibodies capable of specifically binding to the expressed RBP-7 protein or fragments or variants thereof.

[0480] In another embodiment, polyclonal or monoclonal antibodies according to the invention are raised against a RBP-7 polypeptide comprising at least one of the following amino acids:

[0481] a Glycine residue at the amino acid position 293 of SEQ ID No. 29;

[0482] a Glutamic acid at the amino acid in position 963 of SEQ ID No. 29;

[0483] a Methionine residue at the amino acid position 969 of SEQ ID No. 29.

[0484] Alternatively, the antibodies may be capable of binding fragments of the RBP-7 protein which comprise at least 10 amino acids encoded by the sequences of SEQ ID Nos 1 and 4, preferably comprising at least one of the sequences of SEQ ID Nos 30-71 or a fragment or a variant thereof. In some embodiments, the antibodies may be capable of binding fragments of the RBP-7 protein which comprise at least 15 amino acids encoded by the sequences of SEQ ID Nos 1 and 4, preferably comprising at least one of the sequences of SEQ ID Nos 30-71 or a fragment or a variant thereof. In other embodiments, the antibodies may be capable of binding fragments of the RBP-7 protein which comprise at least 25 amino acids encoded by the sequences of SEQ ID Nos 1 and 4, preferably comprising at least one of the sequences of SEQ ID Nos 30-71 or a fragment or a variant thereof. In further embodiments, the antibodies may be capable of binding fragments of the RBP-7 protein which comprise at least 40 amino acids encoded by the sequences of SEQ ID Nos 1 and 4, preferably comprising at least one of the sequences of SEQ ID Nos 30-71 or a fragment or a variant thereof.

[0485] Both monoclonal antibodies and polyclonal antibodies are within the scope of the present invention. Monoclonal or polyclonal antibodies to the protein can then be prepared as follows:

[0486] A. Monoclonal Antibody Production by Hybridoma Fusion

[0487] Monoclonal antibody to epitopes in the RBP-7 protein or a portion thereof can be prepared from murine hybridomas according to the classical method of Kohler and Milstein, (1975) or derivative methods thereof. Briefly, a mouse is repetitively inoculated with a few micrograms of the RBP-7 protein or a portion thereof over a period of a few weeks. The mouse is then sacrificed, and the antibody producing cells of the spleen isolated. The spleen cells are fused by means of polyethylene glycol with mouse myeloma cells, and the excess unfused cells destroyed by growth of the system on selective media comprising aminopterin (HAT media). The successfully fused cells are diluted and aliquots of the dilution placed in wells of a microtiter plate where growth of the culture is continued. Antibody-producing clones are identified by detection of antibody in the supernatant fluid of the wells by immunoassay procedures, such as ELISA, as originally described by Engvall, (1980), and derivative methods thereof. Selected positive clones can be expanded and their monoclonal antibody product harvested for use. Detailed procedures for monoclonal antibody production are described in Davis, L. et al.

[0488] B. Polyclonal Antibody Production by Immunization

[0489] Polyclonal antiserum containing antibodies to heterogeneous epitopes in the RBP-7 protein or a portion thereof can be prepared by immunizing suitable animals with the RBP-7 protein or a portion thereof, which can be unmodified or modified to enhance immunogenicity. Effective polyclonal antibody production is affected by many factors related both to the antigen and the host species. For example, small molecules tend to be less immunogenic than others and may require the use of carriers and adjuvant. Also, host animals vary in response to site of inoculations and dose, with both inadequate or excessive doses of antigen resulting in low titer antisera. Small doses (ng level) of antigen administered at multiple intradermal sites appears to be most reliable. An effective immunization protocol for rabbits can be found in Vaitukaitis, (1971).

[0490] Booster injections can be given at regular intervals, and antiserum harvested when antibody titer thereof, as determined semi-quantitatively, for example, by double immunodiffusion in agar against known concentrations of the antigen, begins to fall. See, for example, Ouchterlony, O. et al. (1973). Plateau concentration of antibody is usually in the range of 0.1 to 0.2 mg/ml of serum. Affinity of the antisera for the antigen is determined by preparing competitive binding curves, as described, for example, by Fisher (1980).

[0491] Antibody preparations prepared according to either protocol are useful in quantitative immunoassays which determine concentrations of antigen-bearing substances in biological samples; they are also used semi-quantitatively or qualitatively to identify the presence of antigen in a biological sample. The antibodies may also be used in therapeutic compositions for killing cells expressing the protein or reducing the levels of the protein in the body.

[0492] Consequently, the invention is also directed to a method for detecting specifically the presence of a polypeptide according to the invention in a biological sample, said method comprising the following steps:

[0493] a) bringing into contact the biological sample with an antibody according to the invention;

[0494] b) detecting the antigen-antibody complex formed.

[0495] Another aspect of the invention is a diagnostic kit for in vitro detecting the presence of a polypeptide according to the present invention in a biological sample, wherein said kit comprises:

[0496] a) a polyclonal or monoclonal antibody as described above, optionally labeled;

[0497] b) a reagent allowing the detection of the antigen-antibody complexes formed, said reagent carrying optionally a label, or being able to be recognized itself by a labeled reagent, more particularly in the case when the above-mentioned monoclonal or polyclonal antibody is not labeled by itself.

Methods For Screening Substances Interacting with a RBP-7 Polypeptide

[0498] For the purpose of the present invention, a ligand means a molecule, such as a protein, a peptide, an antibody or any synthetic chemical compound capable of binding to the RBP-7 protein or one of its fragments or variants or to modulate the expression of the polynucleotide coding for RBP-7 or a fragment or variant thereof.

[0499] In the ligand screening method according to the present invention, a biological sample or a defined molecule to be tested as a putative ligand of the RBP-7 protein is brought into contact with the purified RBP-7 protein, for example the purified recombinant RBP-7 protein produced by a recombinant cell host as described hereinbefore, in order to form a complex between the RBP-7 protein and the putative ligand molecule to be tested.

[0500] The present invention pertains to methods for screening substances of interest that interact with a RBP-7 protein or one fragment or variant thereof. By their capacity to bind covalently or non-covalently to a RBP-7 protein or to a fragment or variant thereof, these substances or molecules may be advantageously used both in vitro and in vivo.

[0501] In vitro, said interacting molecules may be used as detection means in order to identify the presence of a RBP-7 protein in a sample, preferably a biological sample.

[0502] A method for the screening of a candidate substance comprises the following steps:

[0503] a) providing a polypeptide comprising, consisting essentially of, or consisting of a RBP-7 protein or a fragment or a variant thereof;

[0504] b) obtaining a candidate substance;

[0505] c) bringing into contact said polypeptide with said candidate substance;

[0506] d) detecting the complexes formed between said polypeptide and said candidate substance.

[0507] In one embodiment of the screening method defined above, the complexes formed between the polypeptide and the candidate substance are further incubated in the presence of a polyclonal or a monoclonal antibody that specifically binds to the RBP-7 protein or to said fragment or variant thereof.

[0508] Various candidate substances or molecules can be assayed for interaction with a RBP-7 polypeptide. These substances or molecules include, without being limited to, natural or synthetic organic compounds or molecules of biological origin such as polypeptides. When the candidate substance or molecule comprises a polypeptide, this polypeptide may be the resulting expression product of a phage clone belonging to a phage-based random peptide library, or alternatively the polypeptide may be the resulting expression product of a cDNA library cloned in a vector suitable for performing a two-hybrid screening assay.

[0509] The invention also pertains to kits useful for performing the hereinbefore described screening method. Preferably, such kits comprise a RBP-7 polypeptide or a fragment or a variant thereof, and optionally means useful to detect the complex formed between the RBP-7 polypeptide or its fragment or variant and the candidate substance. In a preferred embodiment the detection means are monoclonal or polyclonal antibodies directed against the RBP-7 polypeptide or a fragment or a variant thereof.

[0510] A. Candidate Ligands Obtained Form Random Peptide Libraries

[0511] In a particular embodiment of the screening method, the putative ligand is the expression product of a DNA insert contained in a phage vector (Parmley and Smith, 1988). Specifically, random peptide phages libraries are used. The random DNA inserts encode for peptides of 8 to 20 amino acids in length (Oldenburg K. R. et al., 1992.; Valadon P., et al., 1996.; Lucas A. H., 1994; Westerink M. A. J., 1995; Castagnoli L. et al. (Felici F.), 1991). According to this particular embodiment, the recombinant phages expressing a protein that binds to the immobilized RBP-7 protein is retained and the complex formed between the RBP-7 protein and the recombinant phage may be subsequently immunoprecipitated by a polyclonal or a monoclonal antibody directed against the RBP-7 protein.

[0512] Once the ligand library in recombinant phages has been constructed, the phage population is brought into contact with the immobilized RBP-7 protein. Then the preparation of complexes is washed in order to remove the non-specifically bound recombinant phages. The phages that bind specifically to the RBP-7 protein are then eluted by a buffer (acid pH) or immunoprecipitated by the monoclonal antibody produced by the hybridoma anti-RBP-7, and this phage population is subsequently amplified by an over-infection of bacteria (for example E. coli). The selection step may be repeated several times, preferably 24 times, in order to select the more specific recombinant phage clones. The last step involves characterizing the peptide produced by the selected recombinant phage clones either by expression in infected bacteria and isolation, expressing the phage insert in another host-vector system, or sequencing the insert contained in the selected recombinant phages.

[0513] B. Candidate Ligands Obtained Through a Two-Hybrid Screening Assay

[0514] The yeast two-hybrid system is designed to study protein-protein interactions in vivo (Fields and Song, 1989), and relies upon the fusion of a bait protein to the DNA binding domain of the yeast Gal4 protein. This technique is also described in the U.S. Pat. No. 5,667,973 and the U.S. Pat. No. 5,283,173 (Fields et al.) the technical teachings of both patents being herein incorporated by reference.

[0515] The general procedure of library screening by the two-hybrid assay may be performed as described by Harper et al. (Harper J W et al., 1993) or as described by Cho et al. (1998) or also Fromont-Racine et al. (1997).

[0516] The bait protein or polypeptide comprises, consists essentially of, or consists of a RBP-7 polypeptide or a fragment or variant thereof.

[0517] More precisely, the nucleotide sequence encoding the RBP-7 polypeptide or a fragment or variant thereof is fused to a polynucleotide encoding the DNA binding domain of the GAL4 protein, the fused nucleotide sequence being inserted in a suitable expression vector, for example pAS2 or pM3.

[0518] Then, a human cDNA library is constructed in a specially designed vector, such that the human cDNA insert is fused to a nucleotide sequence in the vector that encodes the transcriptional domain of the GAL4 protein. Preferably, the vector used is the pACT vector. The polypeptides encoded by the nucleotide inserts of the human cDNA library are termed “pray” polypeptides.

[0519] A third vector contains a detectable marker gene, such as β galactosidase gene or CAT gene that is placed under the control of a regulation sequence that is responsive to the binding of a complete Gal4 protein containing both the transcriptional activation domain and the DNA binding domain. For example, the vector pG5EC may be used.

[0520] Two different yeast strains are also used. As an illustrative but non limiting example the two different yeast strains may be the followings:

[0521] Y190, the phenotype of which is (MATa, Leu2-3, 112 ura3-12, trp1-901, his3-D200, ade2-101, gal4Dgal180D URA3 GAL-LacZ, LYS GAL-HIS3, cyh′);

[0522] Y187, the phenotype of which is (MATa gal4 gal80 his3 trp1-901 ade2-101 ura3-52 leu2-3, -112 URA3 GAL-lacZmet⁻), which is the opposite mating type of Y190.

[0523] Briefly, 20 μg of pAS2/RBP-7 and 20 μg of pACT-cDNA library are co-transformed into yeast strain Y190. The transformants are selected for growth on minimal media lacking histidine, leucine and tryptophan, but containing the histidine stnthesis inhibitor 3-AT (50 mM). Positive colonies are screened for beta galactosidase by filter lift assay. The double positive colonies (His⁺, β-gal⁺) are then grown on plates lacking histidine, leucine, but containing tryptophan and cycloheximide (10 mg/ml) to select for loss of pAS2/RBP-7 plasmids bu retention of pACT-cDNA library plasmids. The resulting Y190 strains are mated with Y187 strains expressing RBP-7 or non-related control proteins; such as cyclophilin B, lamin, or SNF1, as Gal4 fusions as described by Harper et al. (Harper J W et al., 1993) and by Bram et al. (Bram R J et al., 1993), and screened for β galactosidase by filter lift assay. Yeast clones that are β gal-after mating with the control Gal4 fusions are considered false positives.

[0524] In another embodiment of the two-hybrid method according to the invention, interaction between RBP-7 or a fragment or variant thereof with cellular proteins may be assessed using the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech). As described in the manual accompanying the Matchmaker Two Hybrid System 2 (Catalog No. K1604-1, Clontech), the disclosure of which is incorporated herein by reference, nucleic acids encoding the RBP-7 protein or a portion thereof, are inserted into an expression vector such that they are in frame with DNA encoding the DNA binding domain of the yeast transcriptional activator GAL4. A desired cDNA, preferably human cDNA, is inserted into a second expression vector such that they are in frame with DNA encoding the activation domain of GAL4. The two expression plasmids are transformed into yeast and the yeast are plated on selection medium which selects for expression of selectable markers on each of the expression vectors as well as GALA dependent expression of the HIS3 gene. Transformants capable of growing on medium lacking histidine are screened for GAL4 dependent lacZ expression. Those cells which are positive in both the histidine selection and the lacZ assay contain interaction between RBP-7 and the protein or peptide encoded by the initially selected cDNA insert.

[0525] C. Candidate Ligand Obtained Through Biosensor Assay

[0526] Proteins interacting with the RBP-7 protein or portions thereof can also be screened by using an Optical Biosensor as described in Edwards et Leatherbarrow (1997), the disclosure of which is incorporated herein by reference. The main advantage of the method is that it allows the determination of the association rate between the protein and other interacting molecules. Thus, it is possible to specifically select interacting molecules with a high or low association rate. Typically a target molecule is linked to the sensor surface (through a carboxymethl dextran matrix) and a sample of test molecules is placed in contact with the target molecules. The binding of a test molecule to the target molecule causes a change in the refractive index and/or thickness. This change is detected by the Biosensor provided it occurs in the evanescent field (which extend a few hundred nanometers from the sensor surface). In these screening assays, the target molecule can be the RBP-7 protein or a portion thereof and the test sample can be a collection of proteins extracted from tissues or cells, a pool of expressed proteins, combinatorial peptide and/or chemical libraries,or phage displayed peptides. The tissues or cells from which the test proteins are extracted can originate from any species.

Method for Screening Ligands that Modulate the Expressions of the RBP-7 Gene

[0527] The present invention also concerns a method for screening substances or molecules that are able to increase, or in contrast to decrease, the level of expression of the RBP-7 gene. Such a method may allow the one skilled in the art to select substances exerting a regulating effect on the expression level of the RBP-7 gene and which may be useful as active ingredients included in pharmaceutical compositions for treating patients suffering from deficiencies in the regulation of expression of the RBP-7 gene.

[0528] Thus, another aspect of the present invention is a method for the screening of a candidate substance or molecule, said method comprising the following steps:

[0529] a) providing a recombinant cell host containing a nucleic acid, wherein said nucleic acid comprises a nucleotide sequence selected from the group consisting of SEQ ID Nos: 1, 4, 30-75 or the sequences complementary thereto or a fragment or a variant thereof;

[0530] b) obtaining a candidate substance, and

[0531] c) determining the ability of the candidate substance to modulate the expression levels of the nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID No: 1, 4, 30-75 or the sequences complementary thereto or a fragment or a variant thereof.

[0532] The invention also pertains to kits useful for performing the hereinbefore described screening method. Preferably, such kits comprise a recombinant vector that allows the expression of a nucleic acid comprising a nucleotide sequence selected from the group consisting of SEQ ID No: 1, 4, 30-75 or the sequences complementary thereto or a fragment or a variant thereof or, alternatively, the kit may comprise a recombinant cell host containing such recombinant vectors.

[0533] Another subject of the present invention is a method for screening molecules that modulate the expression of the RBP-7 protein. Such a screening method comprises the steps of:

[0534] a) cultivating a prokaryotic or an eukaryotic cell that has been transfected with a nucleotide sequence encoding the RBP-7 protein, placed under the control of its own promoter;

[0535] b) bringing into contact the cultivated cell with a molecule to be tested;

[0536] c) quantifying the expression of the RBP-7 protein.

[0537] In another embodiment of a method for screening of a candidate substance or molecule that modulates the expression of the RBP-7 gene, the method comprises the following steps:

[0538] a) providing a recombinant cell host containing a nucleic acid, wherein said nucleic acid comprises the nucleotide sequence of SEQ ID No. 2, the sequence complementary thereto, or a biologically active fragment or variant thereof located upstream a polynucleotide encoding a detectable protein;

[0539] b) obtaining a candidate substance, and

[0540] c) determining the ability of the candidate substance to modulate the expression levels of the polynucleotide encoding the detectable protein.

[0541] Among the preferred polynucleotides encoding a detectable protein, there may be cited polynucleotides encoding β galactosidase, green fluorescent protein (GFP) and chloramphenicol acetyl transferase (CAT).

[0542] The invention also pertains to kits useful for performing the hereinbefore described screening method. Preferably, such kits comprise a recombinant vector that allows the expression of a nucleotide sequence of SEQ ID No. 2 or a biologically active fragment or variant thereof located upstream a polynucleotide encoding a detectable protein.

[0543] For the design of suitable recombinant vectors useful for performing the screening methods described above, it will be referred to the section of the present specification wherein the preferred recombinant vectors of the invention are detailed.

[0544] Using DNA recombination techniques well known by the one skill in the art, the RBP-7 protein encoding DNA sequence is inserted into an expression vector, downstream from its promoter sequence. As an illustrative example, the promoter sequence of the RBP-7 gene is contained in the nucleic acid of SEQ ID No. 2.

[0545] The quantification of the expression of the RBP-7 protein may be realized either at the mRNA level or at the protein level. In the latter case, polyclonal or monoclonal antibodies may be used to quantify the amounts of the RBP-7 protein that have been produced, for example in an ELISA or a RIA assay.

[0546] In a preferred embodiment, the quantification of the PBP-7 mRNA is realized by a quantitative PCR amplification of the cDNA obtained by a reverse transcription of the total mRNA of the cultivated RBP-7-transfected host cell, using a pair of primers specific for RBP-7.

[0547] Expression levels and patterns of RBP-7 may be analyzed by solution hybridization with long probes as described in International Patent Application No. WO 97/05277, the entire contents of which are incorporated herein by reference. Briefly, the RBP-7 cDNA or the RBP-7 genomic DNA described above, or fragments thereof, is inserted at a cloning site immediately downstream of a bacteriophage (T3, T7 or SP6) RNA polymerase promoter to produce antisense RNA. Preferably, the RBP-7 insert comprises at least 100 or more consecutive nucleotides of the genomic DNA sequence or the cDNA sequences, particularly those comprising at least one of SEQ ID Nos 30-71 or those encoding mutated RBP-7. The plasmid is linearized and transcribed in the presence of ribonucleotides comprising modified ribonucleotides (i.e. biotin-UTP and DIG-UTP). An excess of this doubly labeled RNA is hybridized in solution with mRNA isolated from cells or tissues of interest. The hybridizations are performed under standard stringent conditions (40-50° C. for 16 hours in an 80% formamide, 0.4 M NaCl buffer, pH 7-8). The unhybridized probe is removed by digestion with ribonucleases specific for single-stranded RNA (i.e. RNases CL3, T1, Phy M, U2 or A). The presence of the biotin-UTP modification enables capture of the hybrid on a microtitration plate coated with streptavidin. The presence of the DIG modification enables the hybrid to be detected and quantified by ELISA using an anti-DIG antibody coupled to alkaline phosphatase.

Methods for Inhibiting the Expressions of a RBP-7 Gene

[0548] Other therapeutic compositions according to the present invention comprise advantageously an oligonucleotide fragment of the nucleic sequence of RBP-7 as an antisense tool or a triple helix tool that inhibits the expression of the corresponding RBP-7 gene.

[0549] A—Antisense Approach

[0550] Preferred methods using antisense polynucleotide according to the present invention are the procedures described by Sczakiel et al. (Sczakiel G. et al., 1995).

[0551] Preferably, the antisense tools are choosen among the polynucleotides (15-200 bp long) that are complementary to the 5′ end of the RBP-7 mRNA. In another embodiment, a combination of different antisense polynucleotides complementary to different parts of the desired targetted gene are used.

[0552] Preferred antisense polynucleotides according to the present invention are complementary to a sequence of the mRNAs of RBP-7 that contains the translation initiation codon ATG.

[0553] The antisense nucleic acid molecules to be used in gene therapy may be either DNA or RNA sequences. They comprise a nucleotide sequence complementary to the targeted sequence of the RBP-7 genomic DNA, the sequence of which can be determined using one of the detection methods of the present invention. In a preferred embodiment, the antisense oligonucleotide are able to hybridize with at least one of the splicing sites of the targeted RBP-7 gene, or with the 3′ UTR of the 5′ UTR. The antisense nucleic acids should have a length and melting temperature sufficient to permit formation of an intracellular duplex having sufficient stability to inhibit the expression of the RBP-7 mRNA in the duplex. Strategies for designing antisense nucleic acids suitable for use in gene therapy are disclosed in Green et al., (1986) and Izant and Weintraub, (1984), the disclosures of which are incorporated herein by reference.

[0554] In some strategies, antisense molecules are obtained by reversing the orientation of the RBP-7 coding region with respect to a promoter so as to transcribe the opposite strand from that which is normally transcribed in the cell. The antisense molecules may be transcribed using in vitro transcription systems such as those which employ T7 or SP6 polymerase to generate the transcript. Another approach involves transcription of RBP-7 antisense nucleic acids in vivo by operably linking DNA containing the antisense sequence to a promoter in a suitable expression vector.

[0555] Alternatively, suitable antisense strategies are those described by Rossi et al. (1991), in the International Applications Nos. WO 94/23026, WO 95/04141, WO 92/18522 and in the European Patent Application No. EP 0 572 287 A2, the disclosures of which are incorporated herein by reference in their entireties.

[0556] An alternative to the antisense technology that is used according to the present invention involves using ribozymes that will bind to a target sequence via their complementary polynucleotide tail and that will cleave the corresponding RNA by hydrolyzing its target site (namely “hammerhead ribozymes”). Briefly, the simplified cycle of a hammerhead ribozyme involves (1) sequence specific binding to the target RNA via complementary antisense sequences; (2) site-specific hydrolysis of the cleavable motif of the target strand; and (3) release of cleavage products, which gives rise to another catalytic cycle. Indeed, the use of long-chain antisense polynucleotide (at least 30 bases long) or ribozymes with long antisense arms are advantageous. A preferred delivery system for antisense ribozyme is achieved by covalently linking these antisense ribozymes to lipophilic groups or to use liposomes as a convenient vector. Preferred antisense ribozymes according to the present invention are prepared as described by Sczakiel et al. (1995), the specific preparation procedures being referred to in said article being herein incorporated by reference.

[0557] B—Triple Helix Approach

[0558] The RBP-7 genomic DNA may also be used to inhibit the expression of the RBP-7gene based on intracellular triple helix formation.

[0559] Triple helix oligonucleotides are used to inhibit transcription from a genome. They are particularly useful for studying alterations in cell activity when it is associated with a particular gene.

[0560] Similarly, a portion of the RBP-7 genomic DNA can be used to study the effect of inhibiting RBP-7 transcription within a cell. Traditionally, homopurine sequences were considered the most useful for triple helix strategies. However, homopyrimidine sequences can also inhibit gene expression. Such homopyrimidine oligonucleotides bind to the major groove at homopurine:homopyrimidine sequences. Thus, both types of sequences from the RBP-7 genomic DNA are contemplated within the scope of this invention.

[0561] To carry out gene therapy strategies using the triple helix approach, the sequences of the RBP-7 genomic DNA are first scanned to identify 10-mer to 20-mer homopyrimidine or homopurine stretches which could be used in triple-helix based strategies for inhibiting RBP-7 expression. Following identification of candidate homopyrimidine or homopurine stretches, their efficiency in inhibiting RBP-7 expression is assessed by introducing varying amounts of oligonucleotides containing the candidate sequences into tissue culture cells which express the RBP-7 gene.

[0562] The oligonucleotides can be introduced into the cells using a variety of methods known to those skilled in the art, including but not limited to calcium phosphate precipitation, DEAE-Dextran, electroporation, liposome-mediated transfection or native uptake.

[0563] Treated cells are monitored for altered cell function or reduced RBP-7 expression using techniques such as Northern blotting, RNase protection assays, or PCR based strategies to monitor the transcription levels of the RBP-7 gene in cells which have been treated with the oligonucleotide.

[0564] The oligonucleotides which are effective in inhibiting gene expression in tissue culture cells may then be introduced in vivo using the techniques described above in the antisense approach at a dosage calculated based on the in vitro results, as described in antisense approach.

[0565] In some embodiments, the natural (beta) anomers of the oligonucleotide units can be replaced with alpha anomers to render the oligonucleotide more resistant to nucleases. Further, an intercalating agent such as ethidium bromide, or the like, can be attached to the 3′ end of the alpha oligonucleotide to stabilize the triple helix. For information on the generation of oligonucleotides suitable for triple helix formation see Griffin et al. (1989), which is hereby incorporated by this reference.

[0566] Throughout this application, various references are referred to within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the sate of the art to which this invention pertains.

EXAMPLES Example 1 Analysis of the mRNAs Encoding a RBP-7 Polypeptide Synthesized by the Cells

[0567] RBP-7 cDNA was obtained as follows: 4 μl of ethanol suspension containing 1 mg of human prostate total RNA (Clontech laboratories, Inc., Palo Alto, USA; Catalogue N. 64038-1) was centrifuged, and the resulting pellet was air dried for 30 minutes at room temperature.

[0568] First strand cDNA synthesis was performed using the AdvantageTM RT-for- PCR kit (Clontech laboratories Inc., catalogue N. K1402-1). 1 μl of 20 mM solution of a specific oligo dT primer was added to 12.5 μl of RNA solution in water, heated at 74° C. for 2.5 min and rapidly quenched in an ice bath. 10 μl of 5×RT buffer (50 mM Tris-HCI, pH 8.3, 75 mM KCl, 3 mM MgCl₂), 2.5 μl of dNTP mix (10 mM each), 1.25 μl of human recombinant placental RNA inhibitor were mixed with 1 ml of MMLV reverse transcriptase (200 units). 6.5 μl of this solution were added to RNA-primer mix and incubated at 42° C. for one hour. 80 μl of water were added and the solution was incubated at 94° C. for 5 minutes.

[0569] 5 μl of the resulting solution were used in a Long Range PCR reaction with hot start, in 50 μl final volume, using 2 units of rtTHXL, 20 pmol/μl of each of 5′-CCCTTGATGAGCCTCCCTATTTGACAG-3′ (SEQ ID No. 137) and 5′-CGCATTGAAATTCCCACGTCGTATTGCCAG-3′ (SEQ ID No. 138) primers with 35 cycles of elongation for 6 minutes at 67° C. in thermocycler.

[0570] The amplification products corresponding to both cDNA strands are partially sequenced in order to ensure the specificity of the amplification reaction.

[0571] Results of Northern blot analysis of prostate mRNAs support the existence of a major RBP-7 cDNA having about 6 kb in length, which is approximately the size of the longest possible RBP-7 transcript.

Example 2 Detection of RBP-7 Biallelic Markers: DNA Extraction

[0572] Donors were unrelated and healthy. They presented a sufficient diversity for being representative of a French heterogeneous population. The DNA from 100 individuals was extracted and tested for the detection of the biallelic markers.

[0573] 30 ml of peripheral venous blood were taken from each donor in the presence of EDTA. Cells (pellet) were collected after centrifugation for 10 minutes at 2000 rpm. Red cells were lysed by a lysis solution (50 ml final volume: 10 mM Tris pH7.6; 5 mM MgCl₂; 10 mM NaCl). The solution was centrifuged (10 minutes, 2000 rpm) as many times as necessary to eliminate the residual red cells present in the supernatant, after resuspension of the pellet in the lysis solution.

[0574] The pellet of white cells was lysed overnight at 42° C. with 3.7 ml of lysis solution composed of:

[0575] 3 ml TE 10-2 (Tris-HCl 10 mM, EDTA 2 mM)/NaCl 0.4 M

[0576] 200 μl SDS 10%

[0577] 500 μl K-proteinase (2 mg K-proteinase in TE 10-2/NaCl 0.4 M).

[0578] For the extraction of proteins, 1 ml saturated NaCl (6M) (1/3.5 v/v) was added. After vigorous agitation, the solution was centrifuged for 20 minutes at 10000 rpm.

[0579] For the precipitation of DNA, 2 to 3 volumes of 100% ethanol were added to the previous supernatant, and the solution was centrifuged for 30 minutes at 2000 rpm. The DNA solution was rinsed three times with 70% ethanol to eliminate salts, and centrifuged for 20 minutes at 2000 rpm. The pellet was dried at 37° C., and resuspended in 1 ml TE 10-1 or 1 ml water. The DNA concentration was evaluated by measuring the OD at 260 nm (1 unit OD=50 μg/ml DNA).

[0580] To determine the presence of proteins in the DNA solution, the OD 260/OD 280 ratio was determined. Only DNA preparations having a OD 260/OD 280 ratio between 1.8 and 2 were used in the subsequent examples described below.

[0581] The pool was constituted by mixing equivalent quantities of DNA from each individual.

Example 3 Detection of the Biallelic Markers: Amplification of Genomic DNA by PCR

[0582] The amplification of specific genomic sequences of the DNA samples of example 2 was carried out on the pool of DNA obtained previously. In addition, 50 individual samples were similarly amplified.

[0583] PCR assays were performed using the following protocol: Final volume 25 μl DNA 2 ng/μl MgCl₂ 2 mM dNTP (each) 200 μM primer (each) 2.9 ng/μl Ampli Taq Gold DNA polymerase 0.05 unit/μl PCR buffer (10× = 0.1 M TrisHCl pH 8.3 0.5 M KCl 1×

[0584] Each pair of primers was designed using the sequence information of the RBP-7 gene disclosed herein and the OSP software (Hillier & Green, 1991). This pair of primers was about 20 nucleotides in length and had the sequences disclosed in Table 1 in the columns labeled PU and RP. TABLE 1 Amplification Amplification primer PU primer RP Amplicon SEQ ID No. SEQ ID No.  5-124 72 87  5-127 73 88  5-128 74 89  5-129 75 90  5-130 76 91  5-131 77 92  5-133 78 93  5-135 79 94  5-136 80 95  5-140 81 96  5-143 82 97  5-145 83 98  5-148 84 99 99-1437 85 100 99-1442 86 101

[0585] Preferably, the primers contained a common oligonucleotide tail upstream of the specific bases targeted for amplification which was useful for sequencing.

[0586] Primers PU contain the following additional PU 5′ sequence: TGTAAAACGACGGCCAGT (SEQ ID No. 139); primers RP contain the following RP 5′ sequence: CAGGAAACAGCTATGACC (SEQ ID No. 140).

[0587] The synthesis of these primers was performed following the phosphoramidite method, on a GENSET UFPS 24.1 synthesizer.

[0588] DNA amplification was performed on a Genius II thermocycler. After heating at 95° C. for 10 min, 40 cycles were performed. Each cycle comprised: 30 sec at 95° C., 54° C. for 1 min, and 30 sec at 72° C. For final elongation, 10 min at 72° C. ended the amplification. The quantities of the amplification products obtained were determined on 96-well microtiter plates, using a fluorometer and Picogreen as intercalant agent (Molecular Probes).

Example 4 Detection of The Biallelic Markers: Sequencing of Amplified Genomic DNA and Identification of Polymorphisms

[0589] The sequencing of the amplified DNA obtained in example 3 was carried out on ABI 377 sequencers. The sequences of the amplification products were determined using automated dideoxy terminator sequencing reactions with a dye terminator cycle sequencing protocol. The products of the sequencing reactions were run on sequencing gels and the sequences were determined using gel image analysis [ABI Prism DNA Sequencing Analysis software (2.1.2 version) and the above mentioned “Trace” basecaller].

[0590] The sequence data were further evaluated using the above mentioned polymorphism analysis software designed to detect the presence of biallelic markers among the pooled amplified fragments. The polymorphism search was based on the presence of superimposed peaks in the electrophoresis pattern resulting from different bases occurring at the same position as described previously.

[0591] Sixteen fragments of amplification were analyzed. In these segments, 21 biallelic markers were detected. The localization of the biallelic markers is as shown in Table 2. TABLE 2 Marker Localization BM position in Poly- SEQ ID No. Amplicon BM Name in RBP-7 SEQ ID No. 1 morphism Allele 1 Allele 2  5-124 A1  5-124-273 Intron 5 72794 A*/G 30 51  5-127 A2  5-127-261 Intron 8 88073  A/C* 31 52  5-128 A3  5-128-60 Intron 8 93714 Del(GT) 32 53  5-129 A4  5-129-144 Intron 9 97152 Del(T) 33 54  5-130 A5  5-130-257 Exon 11 99098 A*/G 34 55  5-130 A6  5-130-276 Exon 11 99117  A/G 35 56  5-131 A7  5-131-395 Intron 12 103806 A*/T 36 57  5-133 A8  5-133-375 Intron 14 106940 ins(A) 37 58  5-135 A9  5-135-155 Intron 15 108106 ins(A) 38 59  5-135 A10  5-135-198 Intron 15 108149 ins(GTTT) 39 60  5-135 A11  5-135-357 Intron 15 108308 A*/G 40 61  5-136 A12  5-136-174 Exon 16 108471  C/T* 41 62  5-140 A13  5-140-120 Intron 18 134134  C/T* 42 63  5-140 A14  5-140-348 Intron 19 134362 ins(A) 43 64  5-140 A15  5-140-361 Intron 19 134374 ins(CA) 44 65  5-143 A16  5-143-101 Exon 20 146345  A/C 45 66  5-143 A17  5-143-84 Exon 20 146328  A/G* 46 67  5-145 A18  5-145-24 Intron 20 150329 A*/G 47 68  5-148 A19  5-148-352 Exon 24 160031  G/T 48 69 99-1437 A20 99-1437-325 Intron 8 90842  A/G 49 70 99-1442 A21 99-1442-224 Intron 9 97122  G/T 50 71

Example 5 Validation of the Polymorphisms Through Microsequencing

[0592] The biallelic markers identified in example 4 were further confirmed and their respective frequencies were determined through microsequencing. Microsequencing was carried out for each individual DNA sample described in Example 2.

[0593] Amplification from genomic DNA of individuals was performed by PCR as described above for the detection of the biallelic markers with the same set of PCR primers (Table 1).

[0594] The preferred primers used in microsequencing were about 23 nucleotides in length and hybridized just upstream of the considered polymorphic base. According to the invention, the primers used in microsequencing are detailed in Table 3. TABLE 3 Mis. 1 in Mis. 2 in Marker Name SEQ ID No. SEQ ID No.  5-124-273 102 123  5-127-261 103 123  5-128-60 104 —  5-129-144 105 —  5-130-257 106 125  5-130-276 107 126  5-131-395 108 127  5-133-375 109 —  5-135-155 110 —  5-135-198 111 —  5-135-357 112 128  5-136-174 113 129  5-140-120 114 130  5-140-348 115 —  5-140-361 116 —  5-143-101 117 131  5-143-84 118 132  5-145-24 119 133  5-148-352 120 134 99-1437-325 121 135 99-1442-224 122 136

[0595] The microsequencing reaction was performed as follows

[0596] After purification of the amplification products, the microsequencing reaction mixture was prepared by adding, in a 20 μl final volume: 10 pmol microsequencing oligonucleotide, 1 U Thermosequenase (Amersham E79000G), 1.25 μl Thermosequenase buffer (260 mM Tris HCl pH 9.5, 65 mM MgCl₂), and the two appropriate fluorescent ddNTPs (Perkin Elmer, Dye Terminator Set 401095) complementary to the nucleotides at the polymorphic site of each biallelic marker tested, following the manufacturer's recommendations. After 4 minutes at 94° C., 20 PCR cycles of 15 sec at 55° C., 5 sec at 72° C., and 10 sec at 94° C. were carried out in a Tetrad PTC-225 thermocycler (MJ Research). The unincorporated dye terminators were then removed by ethanol precipitation. Samples were finally resuspended in formamide-EDTA loading buffer and heated for 2 min at 95° C. before being loaded on a polyacrylamide sequencing gel. The data were collected by an ABI PRISM 377 DNA sequencer and processed using the GENESCAN software (Perkin Elmer).

[0597] Following gel analysis, data were automatically processed with software that allows the determination of the alleles of biallelic markers present in each amplified fragment.

[0598] The software evaluates such factors as whether the intensities of the signals resulting from the above microsequencing procedures are weak, normal, or saturated, or whether the signals are ambiguous. In addition, the software identifies significant peaks (according to shape and height criteria). Among the significant peaks, peaks corresponding to the targeted site are identified based on their position. When two significant peaks are detected for the same position, each sample is categorized classification as homozygous or heterozygous type based on the height ratio.

[0599] Although this invention has been described in terms of certain preferred embodiments, other embodiments which will be apparent to those of ordinary skill in the art in view of the disclosure herein are also within the scope of this invention. Accordingly, the scope of the invention is intended to be defined only by reference to the appended claims. All documents cited herein are incorporated herein by reference in their entirety.

References

[0600] The disclosures of all of the following publications are incorporated herein by reference in their entireties:

[0601] Abbondanzo S J et al., 1993, Methods in Enzymology, Academic Press, New York, pp. 803-823.

[0602] Adra et al.,1987, Gene, 60:65-74

[0603] Anton M. et al., 1995, J. Virol., 69: 4600-4606.

[0604] Araki K. et al., 1995, Proc. Natl. Acad. Sci. USA, 92: 160-164.

[0605] Baubonis et al., 1993, Nucleic Acids Res., 21: 2025-2029.

[0606] Beard et al., 1980, Virology, Vol. 75:81

[0607] Beaucage et al., Tetrahedron Lett 1981, 22: 1859-1862

[0608] Bolmont et al., J. of Submicroscopic cytology and pathology, 1990, 22:117-122

[0609] Bradley A., 1987, Production and analysis of chimaeric mice. In: E. J. Robertson (Ed.), Teratocarcinomas and embryonic stem cells: A practical approach. IRL Press, Oxford, pp.113.

[0610] Bram R J et al., 1993, Mol. Cell Biol., 13: 47604769

[0611] Brown E L, Belagaje R, Ryan M J, Khorana H G, Methods Enzymol 1979;68:109-151

[0612] Castagnoli L. et al. (Felici F.), 1991, J. Mol. Biol., 222:301-310

[0613] Chai H. et al., 1993, Biotechnol. Appl. Biochem., 18:259-273

[0614] Cho R J et al., 1998, Proc. Natl. Acad. Sci. USA, 95(7): 3752-3757.

[0615] Chou J. Y., 1989, Mol. Endocrinol., 3 :1511-1514.

[0616] Compton J. Nature. 1991 Mar 7; 350(6313): 91-92.

[0617] Current Protocols in Molecular Biology, 1989, Ausubel F M et al. (eds), Greene Publishing Associates, Sections 9.10-9.14.

[0618] Davis, L. et al. Basic Methods in Molecular Biology Elsevier, New York. Section 21-2

[0619] Defeo-Jones et al. Nature, 1991, Vol. 352 : 251-254

[0620] Eckner R. et al., 1991, EMBO J., 10: 3513-3522.

[0621] Edwards et Leatherbarrow, Analytical Biochemistry, 246, 1-6 (1997)

[0622] Feldman and Steg, 1996, Medecine/Sciences, synthese, 12:47-55

[0623] Felgner et al., 1987, Proc. Natl. Acad. Sci., 84:7413

[0624] Fields and Song, 1989, Nature, 340: 245-246

[0625] Fisher, D., Chap. 42 in: Manual of Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc. For Microbiol., Washington, D.C. (1980).

[0626] Flotte et al., 1992, Am. J. Respir. Cell Mol. Biol., 7: 349-356.

[0627] Fraley et al., 1980, J. Biol. Chem., 255:10431).

[0628] Fromont-Racine M. et al., 1997, Nature Genetics, 16(3): 277-282.

[0629] Fuller S. A. et al., 1996, Immunology in Current Protocols in Molecular Biology, Ausubel et al. Eds, John Wiley & Sons, Inc., USA

[0630] Furth P. A. et al., 1994, Proc. Natl Acad. Sci USA, 91: 9302-9306.

[0631] Gossen M. et al., 1992, Proc. Natl. Acad. Sci. USA, 89 : 5547-5551.

[0632] Gossen M. et al., 1995, Science, 268: 1766-1769.

[0633] Graham, 1984, EMBO J., 3:2917

[0634] Green et al., Ann. Rev. Biochem. 55:569-597 (1986)

[0635] Griffin et al. Science 245:967-971 (1989)

[0636] Grompe, M. et al., Proc. Natl. Acad. Sci. U.S.A 1989; 86:5855-5892

[0637] Grompe, M. Nature Genetics 1993; 5:111-117

[0638] Gu H. et al., 1993, Cell, 73: 1155-1164.

[0639] Gu H. et al., 1994, Science, 265: 103-106.

[0640] Guatelli J C et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 1874-1878.

[0641] Guzman, 1981, Cell, 23: 175

[0642] Hacia J G, Brody L C, Chee M S, Fodor S P, Collins F S, Nat Genet 1996;14(4):441-447

[0643] Hames B D and Higgins S J, 1985, “Nucleic acid hybridization: a practical approach”, Hames and Higgins Ed., IRL Press, Oxford.

[0644] Harper J W et al., 1993, Cell, 75: 805-816

[0645] Harris H. et al., 1969, Nature, 223 : 363-368

[0646] Helin et al., 1992, Cell, 70 : 337-350

[0647] Hillier L. and Green P. Methods Appl., 1991, 1: 124-8.

[0648] Hoess et al., 1986, Nucleic Acids Res., 14: 2287-2300.

[0649] Houbenweyl, 1974, in Meuthode der Organischen Chemie, E. Wunsch Ed., Volume 15-I et 15-II, Thieme, Stuttgart

[0650] Huygen et al., 1996, Nature Medicine, 2(8):893-898

[0651] Izant J G, Weintraub H, Cell 1984 Apr;36(4):1007-15

[0652] Julan et al., 1992, J. Gen. Virol., 73: 3251-3255.

[0653] Kaneda et al., 1989, Science, 243:375

[0654] Kanegae Y. et al., Nucl. Acids Res., 23: 3816-3821.

[0655] Kim et al., 1994, Mol. And Cell Biol., 14(11): 7256-7264

[0656] Kim U. -J., et al., 1996, Genomics, 34: 213-218.

[0657] Koch Y., 1977, Biochem. Biophys. Res. Commun., 74:488491

[0658] Kohler G. and Milstein C., 1975, Nature, 256: 495.

[0659] Koller et al., 1992, Annu. Rev. Immunol., 10: 705-30.

[0660] Kort et al., 1983, Nucleic Acids Research, 11:8287-8301

[0661] Kozal M J, Shah N, Shen N, Yang R, Fucini R, Merigan T C, Richman D D, Morris D, Hubbell E, Chee M, Gingeras T R, Nat Med 1996;2(7):753-759

[0662] Lenhard T. et al., 1996, Gene, 169:187-190

[0663] Levrero et al., 1991, Gene, 101:195

[0664] Linton M. F. et al., 1993, J. Clin. Invest., 92: 3029-3037.

[0665] Liu Z. et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 45284262.

[0666] Lucas A. H., 1994, In: Development and Clinical Uses of Haempophilus b Conjugate;

[0667] Mansour S. L. et al., 1988, Nature, 336:348-352.

[0668] Mansour S L et al., 1988, Nature, 336:348-352.

[0669] Manz et al., Adv in Chromatogr 1993; 33:1-66

[0670] Marshall R. L., et al., PCR Methods and Applications 4: 80-84 (1994)

[0671] McCormick et al., 1994, Genet. Anal. Tech. Appl., 11: 158-164.

[0672] McLaughlin et al., 1989, J. Virol., 62: 1963-1973.

[0673] Merrifield RB, 1965, Nature, 207(996): 522-523.

[0674] Merrifield RB., 1965, Science, 150(693): 178-185.

[0675] Midoux, 1993, Nucleic Acids Research, 21:871-878

[0676] Muzyczka et al., 1992, Cur. Topics in Micro. and Immunol., 158: 97-129.

[0677] Nada S. et al., 1993, Cell, 73: 1125-1135.

[0678] Nagy A. et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 8424-8428.

[0679] Narang S A, Hsiung H M, Brousseau R, Methods Enzymol 1979;68:90-98

[0680] Neda et al., 1991, J. Biol. Chem., 266: 14143-14146.

[0681] O'Reilly et al., 1992, Baculovirus expression vectors: a Laboratory Manual. W.H. Freeman and Co., New York Adra et al., 1987, Gene, 60:65-74

[0682] Ohno et al., 1994, Sciences, 265:781-784

[0683] Oldenburg K. R. et al., 1992, Proc. Natl. Acad. Sci., 89:5393-5397.

[0684] Otterson et al., 1993, Oncogene, 8:949-957

[0685] Ouchterlony, O. et al., Chap. 19 in: Handbook of Experimental Immunology D. Wier (ed) Blackwell (1973)

[0686] Pagano et al., 1967, J. Virol., 1:891

[0687] Parmley and Smith, Gene, 1988, 73:305-318

[0688] Pastore, 1994, Circulation, 90:I-517

[0689] PCR Methods and Applications” (1991, Cold Spring Harbor Laboratory Press

[0690] Pease S. ans William R. S., 1990, Exp. Cell. Res., 190: 209-211.

[0691] Peterson et al., 1993, Proc. Natl. Acad. Sci. USA, 90: 7593-7597.

[0692] Porath J et al., 1975, Nature, 258(5536): 598-599.

[0693] Reid L. H. et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 4299-4303.

[0694] Rettlez and Basenga, 1987, Mol. Cell. Biol., 7:1676-1685

[0695] Robertson E., 1987, Embryo-derived stem cell lines. In: E. J. Robertson Ed. Teratocarcinomas and embrionic stem cells: a practical approach. IRL Press, Oxford, pp. 71.

[0696] Rossi et al., Pharmacol. Ther. 50:245-254, (1991)

[0697] Roth J. A. et al., 1996, Nature Medicine, 2(9):985-991

[0698] Rougeot, C. et al.,. Eur. J. Biochem. 219 (3): 765-773, 1994

[0699] Roux et al., 1989, Proc. Natl Acad. Sci. USA, 86: 9079-9083.

[0700] Sakai et al., 1995, Genomics, 30:98-101

[0701] Sambrook, J. Fritsch, E. F., and T. Maniatis. 1989. Molecular cloning: a laboratory manual. 2ed. Cold Spring Harbor Laboratory, Cold spring Harbor, N.Y.

[0702] Samulski et al., 1989, J. Virol., 63:3822-3828.

[0703] Sanchez-Pescador R., 1988, J. Clin. Microbiol., 26(10): 1934-1938

[0704] Sandou et al., 1994, Science, 265: 1875-1878.

[0705] Sauer B. et al., 1988, Proc. Natl. Acad. Sci. USA, 85: 5166-5170.

[0706] Schedl A. et al., 1993a, Nature, 362:258-261.

[0707] Schedl et al., 1993b, Nucleic Acids Res., 21: 4783-4787.

[0708] Sczakiel G. et al., 1995, Trends Microbiol., 1995, 3(6):213-217

[0709] Shay J. W. et al., 1991, Biochem. Biophys. Acta, 1072: 1-7.

[0710] Sheffield, V. C. et al, Proc. Natl. Acad. Sci. U.S.A 1991; 49:699-706

[0711] Shizuya et al., 1992, Porc. Natl. Acad. Sci. USA 89: 8794-8797.

[0712] Shoemaker D D, Lashkari D A, Morris D, Mittmann M, Davis R W, Nat Genet 1996;14(4):450-456

[0713] Smith et al., 1983, Mol. Cell. Biol., 3:2156-2165.

[0714] Sosnowski R G, Tu E, Butler W F, O'Connell J P, Heller M J, Proc Natl Acad Sci USA 1997;94(4):1119-1123

[0715] Stemberg N. L., 1992, Trends Genet., 8: 1-16.

[0716] Stemberg N. L., 1994, Mamm. Genome, 5: 397404.

[0717] Tacson et al., 1996, Nature Medicine, 2(8):888-892

[0718] Te Riele et al., 1990, Nature, 348: 649-651.

[0719] Thomas K. R. et al., 1986, Cell, 44: 419- 428.

[0720] Thomas K. R. et al., 1987, Cell, 51: 503-512.

[0721] Thomson et al., 1998, Science 282, 1145-1147

[0722] Urdea M.S., 1988, Nucleic Acids Research, 11: 4937-4957

[0723] Urdea M S et al., 1991, Nucleic Acids Symp Ser., 24: 197-200.

[0724] Vaitukaitis J. et al., 1971, J. Clin. Endocrinol. Metab., 33:988-991

[0725] Valadon P., et al., 1996, J. Mol. Biol., 261:11-22.

[0726] Van der Lugt et al., 1991, Gene, 105: 263-267.

[0727] Vlasak R. et al., 1983, Eur. J. Biochem., 135:123-126

[0728] Wabiko et al., 1986, DNA, 5(4):305-314

[0729] Walker G. T. et al., Clin. Chem. 42:9-13 [1996]

[0730] Westerink M. A. J., 1995, Proc. Natl. Acad. Sci., 92:4021-4025

[0731] White, B. A. Molecular Cloning to Genetic Engineering Ed. in Methods in Molecular Biology 67: Humana Press, Totowa 1997

[0732] White, M. B. et al., Genomics 1992; 12:301-306

[0733] Wood S. A. et al., 1993, Proc. Natl. Acad. Sci. USA, 90:4582-4585.

[0734] Yagi T. et al., 1990, Proc. Natl. Acad. Sci. USA, 87: 9918-9922.

[0735] Zhenlin et al., Gene, 1989, 78:243-254

[0736] Zou Y. R. et al., 1994, Curr. Biol., 4: 1099-1103.

1 140 1 162450 DNA Homo sapiens allele 72794 5-124-273 polymorphic base A or G misc_feature 1466..1520 homology with EST in ref emblW84531 1 ccccagagta tggactttat ttcccagaaa gccttgaggc gtaactttct gtttccatag 60 aactggtggg aaaatggcgt cgttgtttgt atccagggac caataggaac agtgtatagg 120 cgggttctaa agaactttaa ccaatccaag gtcgtctaag aggccatccg ggaaagaggt 180 aggggagggg gggaaaaaaa atctagggga ggggagaaag ggggggaacc tagagtcggt 240 gggggggaag cgatgtttgc ccgtcagtcg agtccggagt gaggagctcg gtcgccgaag 300 cggagggaga ctcttgagct tcatcttgcc gccgccacgg ccaccgcctg gacctttgcc 360 cggagggagc tgcagagggt ccatcgccgc cgtcctctgg agggcagcgc gattgggggc 420 ccggacctcc agtccggggg ggatttttcg tcgtccccct ccccccaacc agggagcccg 480 agcggccgcc aaacaaaggt accagtcgcc gccgcgggag gaggaggagc cggagcctct 540 gcctcagcag ccgctggacc cgccgccctt cttccccatc tctcccccgg gcctgctggt 600 tttggggggg agaaggagag aggggactct ggacgtgcca gggtcagatc tcgcctccga 660 ggaaggtagg gatattttct ggggctttcg tggtctccta agggggttct tttgggagtc 720 gctgggcccg gccaaggagc agaggaagat cgcggtggtg gtccctgcgg cgcccgaatt 780 cggggcctcg gcctcccggg aacccccacc ccccgcagcc gctgtgtccg agccgccccc 840 tggctgggcg gccgcacact cagcggttta gcggccgcgg tcgggggccc gggcagggtg 900 ggggccgttc cgcctccggg gccctcggcc ctcacgttgt ccccgcggcg gggccagatg 960 ttgatctcgc tcccacttgt cgggtctgag cccggaacgg gacgtgggca ggggctctgt 1020 ggcgggccgg tcctgcccgc ggcccacagg ccctcctggc ccctcggtgg cccccggccg 1080 gcctctcgct cggacgcggc gcgtgggggc gcggattcgc tcggccgggc gccgaggccc 1140 taggggagag cggccggccc tgcgccggac gccgggcttg ttgtgagttt cttctctgac 1200 agaaatggcg tcattgtcgt agacgggaaa ctccgtcggg tctcgacaat ggggacggga 1260 agctgccgag ctgtgtaggt ggttgggttt gcggggatgg cggggccgga gggagccccg 1320 agtcggcgtg tgatggttcg ggggctggcg cctggttgcg gctctttctt cggggttcgg 1380 acgtcgctcg cttctttccc tccgcctccg ccagtgcaga ggggctcctg tggtgttgac 1440 tgggtgtctt ttgtttcccc tccaggtgca gctgaacctg gtgttttaga ggataccttg 1500 gtcccagagt catcatgaag gtaagattgc ctgcggaaaa cgaatctctg gggttatcga 1560 attcaggaag taggaggttt cgacgaggtc gtcgtgtggg gcttccgagt tttgcagagg 1620 aaggtgtgtt tggtgacttg gtgaccgagc ccttccccag agagaccggg gtcagctggc 1680 gggctttgct gtggagttgg aggcagttat aggatgtctc aactgaagat gattctccca 1740 tattgaacat ctctttttgt agggaatgag aggaataacg gcatgaatta aagtgtttta 1800 aaaggtaaaa taaaaagcaa gtcaggtaat ctgaccctag aaactgaact gtattgtggt 1860 tctatcagtt acactgatga aagagaatct tatttattgt gtgaacagta ctgtaaaatt 1920 ttagcagaaa actctttgac tcgtcacaaa aataatttaa ttgaaagatt taaaaagaga 1980 atttttgtga ttttttacac aattcagaga taccagttgt ttcttgtatt tccagtgact 2040 tggggttata tggtttattt ttctttattg atctggttgg tctgtaaata agtcagaggc 2100 tctgttgcat tatgatgtgt ggaaaactga tggccaggtt cctcttagat gtcaatatgt 2160 taactgagtg gagaataggg tcaaaaaatt tgcttaattg ttggaagaaa tgataacgat 2220 aaaaatacag gtatttttgt tacttgagag aatatgtata tgaatgctgt cttttttttt 2280 tttaaacaca tgctagaata aaaccttaga tttttgaaaa gtctggattc aactggtgga 2340 acttagacac tagcttgatt ttttcatcca ttacaaattc agaagtgctt gaaaagggta 2400 gagtgttcac aagtgtagaa atgcaacaga tagcatgtta gaatatcaac gttgtctact 2460 tttgacagtt cctttgaact atttaaaaaa atatttcact tgtcctcaga atcaagactg 2520 ctttgacaat tttgcatcca aattaaaatt tgtaatactt gtggttgtag aaggtaaaag 2580 ctgtggctac ttaaatcttg gtctactata cagagtaatt aagtttagtc taattttttt 2640 aaggttttct ctttgagttt gggatgctgt aaactacctg aaacagttta ttcctatctc 2700 atttcatgaa gcatagacat gtttcaggaa tgaatccctt ttgttagtca caaatcctag 2760 ctataactct catcacctgt gtaaaacagt ggtgaaagtg catttattag gtaactcgta 2820 tatgtacctt gatgttgggt gagaatttct taagaaaagt tttggtttga gtaagaaata 2880 ataagttcag aagccgaata aataaaaggt ttagtttgga gtaaatgtaa tgtgtcacat 2940 ttcccagtgt tgtagtgctt gtcacttctt aggtgctcta taaatgtttg gttaaaagaa 3000 atgtagttaa gttgaaagtc tgagattaaa atgttttttt tcttcctttc gtttcctttt 3060 tttttttaag tagtctgttt aaaaaaatga aagtatgttg gaaactttag ccttaaaagt 3120 ttcttggtaa tactagccaa atgcaaattt tttggtatct ttttagaaat actaatttta 3180 agtgctcagc attacattct actgcatttt ttaaaaaggt gcttttaacg ttaaaaggaa 3240 aaagttacta aaataaattt ctaacttcat acaagtactt cctgttatta taagtatagc 3300 aaatctcaga gaatttgcta gtaattatta gtactaaaaa cttagcttat ttcttacaac 3360 tttatttctt atgtgataaa agatgagaag cttcttgtaa tttgtatttt gctttagcaa 3420 aaattgcaat tcagaatcta tttaatgttg agtgtattag gcctaaattt tgtttctttt 3480 tctgtgtgtt cccaatttct ttcgttcttt tttatttttc tttttgatat agagtctcac 3540 tatttcaccc tggctggcac gatctgtaac tgctgcctcc tgggctcaaa taatcctccc 3600 acctcagcct cctgagtagc tgggactaca ggcacacctg gctaattttt gtagtttttg 3660 tagagatgag gttttgccgt gttgcccagg ctggtgtcga acttctgggc tcaggtgact 3720 ctcccgcctt agcctcccaa agtgctggga ttacaggcga gagtcaccgt gcctggtgta 3780 atttctgtat tacgatgatc attgagctac agaaggtttt atgccattta ttggatctta 3840 ccaaacatat ttgtacttgg gagcaagctt ttaagtttga attgtctgtt gtattaacga 3900 caacaaatct ttaaatttta ttaagtgaca gtttattgag gtgattgaaa gttagattat 3960 gtagtaatac tttttaggtg ttgttttaaa gttgaagtgt tgtatgtttt agcagttttc 4020 ccatgtacta cctttaagaa tttatgtaaa caaatacatt cccattactg tctaatatat 4080 gctatgttac ttttttcaca gtatgttttt gaggaatatg attcgtaata cataaggaat 4140 ttgtgctgta acatagatcg tgatgttggt gttatgttga atttattttt catgctcttg 4200 atagactttt aaaaatcagt ggtgccggct gggcatggtg gctcacgcct gtaatcacag 4260 cactttggga ggccgaggta ggaggatcac ctgaggtcaa gggagtttga gaccagcctg 4320 gccagcgtag taaaaccctg tctcttctaa aaatacaaaa ttagctgggc atggtggcac 4380 acacctgtaa tcccagctat tcggaaggct gaggcaggag aactgcttga acccaggagg 4440 cggaggttgc agtgagctga ggttgcgcca ctgaactcca gcctggcgaa aaaagtgaaa 4500 ctctctctct caaaaaaaaa aaactaaata aaataaaata aaaatcagca gtaccacatt 4560 ataaggaggt ggtagattat taaagtttgt aaataattcc aagacttatt tttattatgt 4620 caggttgatt tttacttttt ttttttaaga cggacttttg ctcttgttgc ccaggccgga 4680 gtgcagtggt gcgatctcgg ctcactgcaa cctctgcctc ccaggttcaa gcagttctcc 4740 tgcctcagcc tcctgagtaa ctggaattat aggcatgcac catcatgccg ggctaatttt 4800 gtgcttttag tagagacaag gtttctccat attggtcagg ctggtctcga actcctgacc 4860 tcaggtgatc tgcccgcctc ggcctcccaa agtgctggga ttacaggcgt gagccactgc 4920 gcccggccga tttttacttt ttttaaactc aaactttttt ttcctgtatc tatattgtac 4980 atgttattaa cgtcttttat tacgtagcag taaattttca attaaaatgg gctggttggg 5040 ctggatatac cagaatcttt agaggaggaa tgatttagat gattatactt tggcgggcat 5100 ggtggctcat gcttgtaatc ccagtacttt gggaggctga ggtgggtgga ttacttgagg 5160 ttgggagttc aagaccagcc tggccaaagt gatgaaaacc ccgtctctac taaaagtata 5220 aaaaaattag ctgggcatgg tggcgcatgc ctgtattccc agctactcag gagactgagg 5280 taggatcgct tgaacctagg aggtggaggt tgcagtgagt cgagatggca ccactgcact 5340 ccagcttggg tgacagggag accccatttc aggaaaaaaa aaaaaaaaag gtggttatat 5400 ttagaatcca cgtagattga attttccaga tgtacttttc tttcttcaac aatgattggc 5460 ttaggccaca tgaaaaaata atggattgat tacaaattcc cttttttaat gccctgtgtg 5520 tgtgtacatg ttctctctct ctctccccct acctctacct ttgtttgtct ccctatccac 5580 tctgtcagtt cgttcccccc tctctgttct tttccttccc cctcttcctt ctgtctcacc 5640 cctttcataa gtcattcatt taacaacaac ccgtttcata tatcaggtaa tatagaatga 5700 aggtgggtga ggcatctgac ctgctttcag ggagcccaca gtgtagcaga agatgcagat 5760 aaataaagca aaactcctag catatctatt tatgagtatt ccaacataaa ataattgtag 5820 gaaatgctat gagaggacag aagaggacag ctaaatcaga gagctatgga aagttttctg 5880 gtggagttga catctaagct gagtgcgaag gcttttgcta ggcaaagaag ggaagaaaag 5940 tttttcagat agaggataac gtactggaaa atgaacgaaa caaggtgaag tatctggaaa 6000 ctgtaagtaa tttggtgtga ctgaattaac ttaaagcatg atatgtttgg gtaggaataa 6060 aagggcagtg gtgggagatg aagctgagtg agggagtcga aaattttctt ggccaggcat 6120 ggtgtctcac acctgtaatc ccagcacttt gggaggttga ggcaggtgga tcacctgagt 6180 tctataccag cctggccaac atggtgaaac cctgtttcta ctaaaaatac aaaattagca 6240 gggcatggtg gtgtgagcct gtaatgccag ctattaggga ggctgaggca gaagaatagc 6300 ttgaacacgg gaggtggagg ttacagtgag ccgagatcat gccattgcac tccagcctgg 6360 gtgacaagag caatactctg tctcaaaaaa aaaaaaattt tttcttgaaa actacatgga 6420 accattgaaa gttgttgaga attgcttgaa cccaggagct ggaggttgcg gtgagctgag 6480 atcaggccac tgcaaaaaca aggggagagc tgaatgtaag gatggaaatt ccaggtaaga 6540 agtccaagtg gaaaatgaat aagggcctga actaatgcat tggaagtgga gcagagagga 6600 tagatctgag cctttttgaa gtagaattca gggccgggca cagtggctca cacctgtaat 6660 cccagcactt tgggaggctg aggtgggtgg atcatgaggt caggagattg agaccatcct 6720 ggctaacaca gtgaaacccc gtctctacta aaaaaataca aaaaatagaa attagccagg 6780 cttggtagcg ggcgcctgta ggacgctgag gcaggagaat ggtgcgaacc tgggaggcgg 6840 agcttgcagt gagccgagat cacgccactg cactccagcc tgggcgacag agcaagagtc 6900 tgtcttaaaa gaagtagaat tcaggaccta gtaactcatc ctggggtagg aagaagatgg 6960 aggttttttt cttaggaaat tagatagatg ttactgtcat ttgaagcatg ctgtcattgg 7020 acctgctagt taagatgtaa gagtaagtag gtaggatgaa gtcagaggct tggttaatct 7080 ggtgtgaatg ttaacctcat atctatgata tttgaaggga tttaagatcc ataacatggc 7140 aaattttgtg ctataaattt gtggtccttt agatgaatag gtaatcttgt aaaatactaa 7200 catgttttct gtccaaattg ctaaatcttt gtttattgta attaaaaaaa atactaggtg 7260 agtacctatg tcactgaaga atgtttttgc caagtcagcc agtgaattca gtcattgaac 7320 acattgtgta ctttccttgt accggacatt gttctgggtt ttgaggatgc agcagtgaat 7380 agaacaagag ttcttaccct cgtggagttt actttctact atggtggaca cacgttaaga 7440 aaaaaaataa tgctaagtgc tttgaagaga aatgatacgg aatcagaata agaggttaga 7500 gtgacagagg gtgggttact ttagattagg tgataatgga gggccttttc gagatgacat 7560 ttaagtgtgc tgtgaaacaa gtgatagagt gagccattca ggtttctggg ggaggagcat 7620 tccctgcaga aaacaagtgc aaataccctg agatggagtg tgcttggcat gtttgagtgt 7680 gatacattta aggaacatta aggccatcag ggctggagtg gtaggagagg aggtcagaga 7740 gggtcacaaa gggccagtac aattgtaggg ccttatagga catggtaagg acttgagtaa 7800 aatctgaatg tgttaggaac cctttgaagg gtgttgagtg aagtgatgtg ataaacattt 7860 tcaaaaatgt ttgactgctg ggtagtgaac acatcatggg ttcgaacaga tcatgggaac 7920 acaagagtgg aagtagggag atcagtcaga aggctgtcaa gctagtcctg gggagagatg 7980 gtggtgagga gaaatagtag tattgggaat atatttgaag atggggatga ttggatttca 8040 atggattggg tgtgaaggga aaattctact tactgcaact tgcacatgta tgtcctccca 8100 caagtgccct ccctcctttt cttttgagac ggagcctctc tctgttgcct aggctggagt 8160 gcagtgatgt gatctcggct cactgcagcc tctgtctccc gagtttaagt aattctcttg 8220 ccccagactc ccctgtagct gggattacag gcacctgcca ccacgcctgg ctaatttttt 8280 ttgtattttt tgtttttttt tttttagacg gagtttcgct cgttacccag gttggagtgc 8340 aatggcacga tctcggctca ctgcaacctc tgcctcccag gttcaagcga ttctcctgcc 8400 tcagcctccc aagtagctgg gactacaggc atgtgccacc atgtctggct aatttttgta 8460 tttttagtag agacggggtt tcaccatgtt ggccaggctg gtctcaaact cctgaccttg 8520 ttatccacct gcctcggcct cccagagtgc tgggattaca ggtgtgagtc acctcgcccg 8580 gccttttttt ttgtgttttt agtagcaatg gggtttcacc atgttggcca ggatggttct 8640 gaactcctga ccttaagtga tccgcccgcc ttggcctccc aaagtgtgag ccactgcacc 8700 tggccgtcct cccttttttt aaaaaagtct ttttgctgtg attagttggt agatgggatt 8760 cttggaactg agtatgacca tgaaggcatt gtcaaatctt agccttctca gtgagcagtg 8820 aacattgagt tgagcaatcc tttcacttgt ttttttaatt aaaaaaaatg agatataatt 8880 cacataccat aaaattcacc cttttatttt attttttttg agacatggcc acagtctgtt 8940 gcccaggcag gagtgtagtg gcgcaatcac agctcactgc agccttgatc ttcagtgggc 9000 tcatgtgatc ctgcttcaac ctcccaggta gctgggacca gaggtgcacg gcaccatgcc 9060 cagctaactt ttaaattttc tgtggaggtc tcactgtgtt gcccaggctg gtcttgaact 9120 cctgggctca aatgatcctc ccatcttggc ctaccaaagt gttgggatta caggcatgag 9180 ccaccgcgcc tcaccaaatt caccttttta aagtgttcat aaatttctag tattttcaca 9240 agtttgtgca tccatcacta ctatcttttg aaaacttttt tgttaatttc ttttgtttgt 9300 ttttttagag atgaggtctt gctgtgttac ctaggctggc cttgaactct tgtggtcaag 9360 tgatctttcc ccctcaactt cctgagtagc tgagacttca ggcacgttac ttatccctag 9420 ctatctgtca ctgctattta actccagaac atttccaaat tcccaacaag aaacttctta 9480 tccattagca gtcattcact gttctccctt tcttttatcc ctccagtaac taatctaaat 9540 tttttttttg agaagagttt tgattctgtc acccaggctg gagtgcagtg gctcgaccat 9600 ggctcactgc agcatcgacc tcctgggttc aagcgatctt cctgcctcca gctcccaagt 9660 agatgggatt acaggtgtgc gccaccatgc ccagcaaatt ttggaatttt ttgtagtagt 9720 gaagtctcac tatgttccct agcctggtct ggaactcctg agctcaagtg atcctcttgc 9780 attggccccc aaagtgctgg gattacaaga ttcagacact gagcttagca tttactttct 9840 gtctataagg atttgcgtat tctgggcatt ttctgttaac ggaatcctgt aatacatatg 9900 tggccttttt gtgtctgatt tcttttactt cccataagtg aaagaagtag caagtgttga 9960 ccaggcgcga tggctcacgc ctgtaatccc agcactttgg gaagccgatg tgggtggatc 10020 acgatgtcag gagttcaaga ccagcctggc caagatggtg aaaccccatc tgtactaaat 10080 aaaaaaatta gccgggtgtg gtggtgggcg cctgtaatcc cagctactca ggaggctgag 10140 gcagagaatt gcttgaacct gggaggcaaa ggttgcagtg agctgcgatg gcgccactgc 10200 actccagcct aggtgtcaga tcaagactcc gtctcaaaaa aaaaaaaaaa aaaaaaaaaa 10260 aggagtagca agtgttaata ctatgctctt ttttattgct gaataatagt ccatcttatg 10320 aagatacgac attttgtcag tctacttatt tggaggacat gtgggttatt tctgcttttt 10380 tgcttattgt gagtaatgct gctatgagca tttgtatgta agtttctgtg cgaacatgta 10440 tttttaattc tcttgggtcc tgggagtgga cctttcagtt cttagggtac taatattacc 10500 tgtgtgataa cctggggagt gctgagtatt taacttcatg tattttgtgt tgtggcaagt 10560 ggccaccaag gagttggaca tttaaaaaat taattcaata tttattgaat acttattatg 10620 tattggtctc tgttctagtc agttggaata tgtcagggaa caaaacaggt agaaattcct 10680 ggctctttgg atctcttagg agtacaggaa actctggtat tcaaggtttc cgtagaaaat 10740 aatgtgcttt tagaaatcat aattttcgtt ctaaaattgg agacagcatt cagatttggg 10800 gaagaaaaaa atgagtttat ggaagtatat aaacagttta ctagaaaata aaaagcaggt 10860 ctactaaggt cagcatagac aaactatctt catgttcacc ttaccatagt ggctgttatt 10920 ttaagctggt taatagaagc aagggacata atgaaagagt caaaaaggga aaagttttaa 10980 aacaatactt ggcttactct gagcatagtt tcccctttcc ttaattaacg cttgctcata 11040 tattactcag aaagtccaag tgtaggcttc agaggagagg agccatagat agctttcatc 11100 agattcttga tgaaacccac agtacaaatg ttaagaaaca cagctctgtg gtatcatctg 11160 ttgactgatc tgctgctaat ctatttaata ggaagagttg tttctatatc cttctacttt 11220 taccattaaa gaaaaagtaa tcaactagtc actgttcatt ttattttcaa atttattttt 11280 gcttaaatca ttgcagaatc agaaaaaaat ttttattata ttgtttctga aatgttaaca 11340 tttaggtgaa atgcttaatc aggttgagta tcacttacct gaaatgcttg ggaccagaaa 11400 tatttgggat tttttcagat tttggaatat ttgcatttat atgcttagta tttgaacatc 11460 ccaaatctga aaatccaaaa tgttccagtg agcatttccc tttggtgtaa catgaacact 11520 gaaaaagttt cagtttttgt agcatttctg attttttgtg ttttacgtat gtatatgtat 11580 atctgtatct tgtttttttg tttggttgtt tgagacagaa tcttgctctg tcacccaggc 11640 tggagtgcag tggtgcgatc tcagctcact gcaacactcg gctcccggat tcaaacgatt 11700 cttctccctc agcctcccaa gtagctggga ttacaggcgc ctgccaccac acctggctaa 11760 tttttgtatt tttagtacag acgaggtttc gccatgttgg ccaggctggt ctctcgaact 11820 ccagacctca ggtgatcctc ctgcctcagc ctcccaaagt gctggaatta taggcatcag 11880 ccaccgtgcc cagccttata taaatatatt attatttttt tgagacgaag tcttgctgtg 11940 tcgcccaggc tggagtgcag tagtgcgatc ttggcttact gcagcctctg gctcctgggt 12000 tcaagtgatt ctcctgcctc agcttccaga gtagttggga ttacaggcgt gtgccacaac 12060 acctggctaa ttttttgtat ttttagtgga gacgaggctt caccatgttg gccaggctgg 12120 tctcgaactc ctgacctcaa gtgatccgcc cgcctcagcc ttccaaagtg ctggaattac 12180 aggtgtgagc cactgcaccc cacttatttt tgagttaggg atactcaatg tgaattgctt 12240 gaaatgttta cctcgttgaa ttcctaagaa gaatttgaat tttttaaatt tataactagc 12300 ctttgatcca tggaaacatt ttataaaata atttccaaaa taatttcctg gaaatctgga 12360 attgtagtct gtagcaaatt gggattattt attaatttaa tttaatttaa tttatgagat 12420 cagagtcttg gtatgttgcg ttggctggtc tcgaactcct aggcttgagt gatccttctg 12480 cctcagcctc tctagtggct ggaactgtaa gtgcacacca ccatggcaca ttggatatta 12540 tttatgaaac tatttattac aaatgttagt atatgcttac tcttaccttt tgcatattca 12600 attatttact ctaatcgggt tatctaaggc aagaatagta tctaactgtg aataaccaga 12660 tatgcttagc tttaggatac agttagacgt aagtatagaa ttcaacatcc ctgtaactaa 12720 tgtctttcca gattaatgtt agtgttgata gtaaggtggt agaacgggct aattctctgg 12780 gccattatta ctgatttata aggtagaaaa tagggtgtat cactttaaag ttacaaattt 12840 acatttataa ggaagaaaat agggtatatc acttttaagt tacaaattta cctgtcatca 12900 attaagagaa taataattag gcagtaggtt tataccatta aaatgtgtga gattacttac 12960 actatatctt ggacagtggg acagataatt tatttttttg gagacatagc cttgttctgc 13020 ggcccaggtt ggagtgcggt ggcgcaatca tagctcactg cagcctccag ctcctgggct 13080 caagtgatct tcctacctca gtcacccaag tagctgagac tacaaataat gcacaccacc 13140 atgcctcgct aaattttttt gtatttttgg tagagacggg gtctccttat cttgcccagg 13200 ctagtcttga actcttgagc tcaggtgatc ctcccacctt ggcctcccaa aatgttggga 13260 ttacagatgt gagccaccaa gcctggcctg gacaatggga cagatacttt atatgtagac 13320 ttttctcatt taagccattt ttctctagtt tatagataaa tttttggcaa tgtgacaact 13380 agaatatctg aaatcctata taaaagttct attaatgcta tctgctaatt gataaccttt 13440 tgatttcagg gtataattgc ttatgagaac atagtcattg ataactttag taagtttcat 13500 tgaacatcat attttaacaa tgcacttcaa tagcaaatga agattataaa ctaaaacctt 13560 tgagcaagtg gtatttaaag aaaggattta ctttatattg atagccaaaa taatgtatat 13620 accgaaatat atataaatac gtatttataa acatatttta ctgatagtaa aatgttatat 13680 atattgttat atatcattta agataattta tatgtaaatt aaatataaac cagatttctt 13740 tattccagga accttgctgt tgtttctaac ctttcttttc ctgctactta gtgatgcaga 13800 agcttttctt ggtaaatcta cttgtcccct ctcttaattt actgcttgaa tatgactgtg 13860 aaaactgtct ttgtttaaag tgcccagtaa acattgtagc ttcatgatta agagcatatg 13920 gcttggaaag tcagtggtct tatttccagt cctttttatg tcctttactt ggctgctgag 13980 gtttgggtaa agttactgtt tttgaaatga aagtaaacat cagtttcaat ttttgtaaaa 14040 tgagaataat aacagctacc ttgtaaagtt gtgtaaatat gaaatgaaat accatattta 14100 aaatgcttta cactgtgctt ggcatagttc tgagcagtta gtacgtggca gttgtattat 14160 tagaggaagc ctgtcttgtt tttttttaaa taagctgata gagtgaggat tcttttaatc 14220 aagactgttt gggattgaat tgccactcct gcttaccaga gtgtaggcag tttttcttaa 14280 actttccaag aagactggtg tcctcatcta aaatacgaaa tgcttacagt aattgcctca 14340 tggggttgtt tggggtgact aaatgtagta ggatttacta catagtaagt tctcaataca 14400 ttgtagctat tattattagt tcggtagaaa gaatgtgcag attcttatga gtttaagtag 14460 gctttcgggg agatagattg actctggtct tttaaaagtt aattttgaag ttgcagtttt 14520 gtgattaagc cttaaatctg ttattctttc cttctgaaat ccttaaaaac agaatgttta 14580 gtagaaggtg ataaccagat ttctttattc caagaactct ttgctctcat gtctaacctt 14640 tattttcctg gtacttactg atgcagaagc ttctcttagt aaatataata catctcctct 14700 ctcctaattt gctcccgtct ttccttgtaa gggaaaagta aattttactt ccaagcctag 14760 agggttattt atggattagg tgaactactg aagatactta ttttctggat aagcatccat 14820 ctgtatagcc ttttatgtat ggcaaaaatt gttttcattt cttgatcaga atactgttct 14880 gatgtggtgt agtcagccac ctgaagctga tctagcatgg gcagcctagg caggtagggc 14940 gaatgactgt aggagccctg ctaaacccga gtctctactc cagagaggag ttaaaaaaag 15000 ctgaacaagc ctgaacacgg aggagccact attgctgtca aagttaagtg aagcagcttg 15060 gcttatgtct atttcagaat aaaaaaaaaa aattcaactt aggcatgcac ggtgggtcat 15120 gcccgtaatc ctagcacttt gggcgtttga ggctggcaga ttacttgagg tcaggagttt 15180 gagaccagcc tggccaacat ggtgaaacct tgtctctact gaaaatataa aaattagcca 15240 ggcatggtgg tatgcctgtt tgggaggctg atgcaggaga atctcttgaa cccgggaagt 15300 ggaaattgca gtaagctgag atcgcaccac tgcactccag cctaggtgat agaatgagac 15360 tgcatctcaa aaattcaact tttcacattt tcagttttat cttgataagg atacctcatc 15420 aaagaaccct tttcttttct tttcttttct ttttcttgaa gcagaatctt gctgtgcatc 15480 ccaggctgga gtgcagtggt gcaatcttgg ctcagtgcaa cctccgcctc ctaggttcaa 15540 acgattcttg tgcctcagcc tcctgagtag ctaggactac aggcatgcgc caccatgctc 15600 ggctaatttt ttgtatttta gtagagatgg ggttttgtaa tgttacctag ggtggtctca 15660 aactcccgag ctcaggtgat cgcctgcctt ggcttctcaa agtgctagga ttacaggtgg 15720 gagccaccac gcccagcccc atttctgttt tttttaatct gaatattctt tgctaaagtg 15780 ttgttgtttt ttttaaaggc aagccagaga gcttttggca tttagtaagt cacctattcg 15840 tgctttgctc taacttggag aaatattttt ctaaactagt tcttctagca aaattaaaga 15900 aatttttatt atgaaagcaa taaagatttt ctgtaaacat taaaaattac atagtagtat 15960 tcagtggaca gtagaagtct tacttcctgg taacttgatt caacagtttt tgggtgtatt 16020 cttccacacc tttttctttg cacatatgaa tcaaacatga gtatttattt tgttatgttt 16080 gactgtattg aaatgggctt atgctatgca tattgttcgt aattttcttt tcttttcttt 16140 tctttgagac ggactgtcgc tctgtcacca gactagagtg tggtggcacg atcttggcta 16200 actgcaacct ctgcctcccg ggttcaagca gttctcctgc ctcagcctcc tgagtagctg 16260 gaactacagg tgcgcgccac cacgcccagc taatttttgt atttttagta gacagggttt 16320 caccatgttg gccaggctgg tcttgatctc ttgagctcgt gatttgcctg cctcggcctc 16380 ccaaagtgct gggattacag acgtgagcca ccatgcctgg cccataattt tcttttacat 16440 tcttctatat cagtacatat tacacaagtc tgtgtcattc ttcttgatgg ctttagtaag 16500 attttatttg cattcaacat gttatttttt ctacaaccaa ataatttata tagggcaccg 16560 gtatgtttct ttaagttttg caggtatgta gcagtgctta gtattcagta ggtactgttt 16620 ttgtttgttt gtttgtttgt tttgtttttt gtttttgaga cagggtcttg ctctgttgcc 16680 caggctggag tgcagtggca cagtcttggc tcactgcaac ctctgcctcc cgggttcaag 16740 agattctcat gtttctgcct cccaagtagc tgggactaca gtcccgtgcc atcacaccca 16800 gctaattttt gtatttttat tagagacagg gttttgccac gttggccagg ctggtctcaa 16860 actcctgacc tcaagtgatc tgcctgcctc gacctcccaa agtacaggtg tgagccactg 16920 tgcctggcct tcagtaagta ttgttaactt atttggatgc ctagtactag taggtggcaa 16980 atagtcccag tacctattat atatacaaag cttcttatag tcctatgaat tattattatt 17040 attattatta ttattattat tattattatt attgttttga gacagggtct tgctctgtca 17100 cccaggctgg agtgcacagt acaatcacgg ctcactgtag ccttgacctt ctgggctcaa 17160 gcgatcctcc cacctcagcc tctcgagtag ttgggactac aggtgtgggt caccataggt 17220 tgatttttta tattttctag agatggggtc tcactatgtt gcctaggctg gtcttgaact 17280 cttgccctgt gaattattgc agccaccaac tgttaaatat cattgcatga cattgttact 17340 aaaaggtaat ctatgaggat tagtgaggga gcatccctgt gctatatggc tggttctaaa 17400 aaagcttatg ctgttctttg ggatccctgt tagcattgat tagacaggtt aattttgggg 17460 gccgggtgcg gtggcttata cctgtaatcc cagcagtttg gtaggctgat atgggtggat 17520 tacttgaacc caggagtttg agaccagcat ggacaatgtg gcaaaaccct gcttctacaa 17580 aaaagtttta aaatagccag gagtggtggc ctgtgactgt atttccagct actcaggaag 17640 ttgaggctgg gaggattgct tgaacccagg atgtcaagtc tgcagtggag ctgtgattgc 17700 agccactgta ctccagccta ggtgacagag caagaccccg ttttaaaaac aaaatcaaaa 17760 acacgttaat tttggaatgg atctctatag gaagtgtccc cagcatatgc tcaaagtcag 17820 aatatatagt ttataaggaa ttctttaacc gtacagttat atggcacatt acgtttttaa 17880 agttccataa tcattagtta tatctaataa catccctttg aggcaggtca gcaacatttt 17940 tcccttttta tggtcgaaga agcaggtgta aagatgttga gtgatttgcc cacaggcaca 18000 cattaaactg atcacagagc ctgttctttt gttagtaaac tgaaccatgc tgcctctcta 18060 ctagttatta taaataaata ataaagttgt tgccatttag tgactttttg atggctttat 18120 tgaatagtaa gtgtagttta caaacctttt gcctacttac tttgttgaaa aagttttaaa 18180 tttagaattt acttgtatca tggtatgaaa catttttatg taaatttccc tgtttctttt 18240 tttttatttt tttttattga tcattcttgg gtgtttctca cagaggggga tttggcaggg 18300 tcataggaca atagtggagg gaaggtcagc agataaacaa gtgaacaaag gtctctggtt 18360 ttcctaggca gaggaccctg tggccttccg cagtgtttgt gtccctgggt acttgagatt 18420 agggagtggt gatgactctt aaggagcatg ctgccttcaa gcatctgttt aacaaagcac 18480 atcttgcacc tcccttaatc catttaaccc tgagtggaca cagcacatgt ttcagagagc 18540 acagggttgg gggtaagtaa ggtcacagat caacaggatc ccaaggccga agaatttttc 18600 ttagtacaga acaaaaggaa aagtctccca tgtctacttc tttctacaca gacacggcaa 18660 ccatctgatt tctcaatctt ttccccacct ttcccccctt tctattccac aaaaccgcca 18720 ttgtcatcat ggcccgttct caatgagctg ttgggtacac ctcccagacg gggtggcggc 18780 cgggcagagg ggctcctcac ttcccagtag gggtggccgg gcagaggcgc ccctcacctc 18840 ccggacgggg cggctgaccg ggcggggggc tgaccccccc acctccctcc cggacggggc 18900 ggctggccgg gcagaggggc tcctcacttt ccagtagggg cggccgggca gaggcgcccc 18960 ttacctcccg gatggggcgg ctggccgggc ggggggctga cccccccacc tccctcccgg 19020 acgggtggct gccgggcaga gacgctcctc acttcccaga cggggtggtt gccgggcgga 19080 ggggctcctc acttctcaga tggggcggct gccgggcgga ggggctcctc acttctcaga 19140 tggggcggct gccgggcgga ggggctcctc acttctcaga tggggcggtt gccaggtgga 19200 gggtctcccc acttctcaga cggggcggcc gggcagagac gctcctcacc tcccagacgg 19260 ggtcacggcc gggcagaggc gctcctcaca tcccagacgg ggcggcgggg cagaggtgct 19320 ccccacatct cagacaatgg gcggccgggc agagacgctc ctcacttcct agatgggatg 19380 gcggccggga agaggcgctc ctcacttcct agatgggatg gcggccgggc agagacgctc 19440 gtcactttcc agactgggca gccaggcaga ggggctcctc acgtcccaga cgatgggcgg 19500 ccaggcagag acgctcctca cttcccagat ggggtggcag ccgggcagag gctgcactct 19560 cggcactttg ggaggccaag gcaggtggct gggaggtgga ggttgtagcg agccgagatc 19620 acgccactgc actccagcct gggcaccatt gagcactgag tgaaccagac tccgtctgca 19680 atcccggcac ctcgggaggc tgaggctggc ggatcactcg cggttaggag ctggagacca 19740 gcccggccaa cacagcgaag ccccgtctcc accaaaaaaa tacaaaaacc agtcaggcgt 19800 ggcagcgcgc acctgcaatc gcgggcactc ggcaggctga ggcaggagaa tcaggcaggg 19860 aggttgcagt gagctgagat ggcagcagta cagtccagct tcggctcggc atcagaggga 19920 gaccgtggaa agagagggag agggagaccg tggggagagg gatagggaga gggaggggga 19980 gggggagggg agcccctgtt tcttaaaaat tagaaaaaat caggctggga gcggtggctc 20040 atgcctgtaa tcccagcact ttgggaggcc aaggtgggcg aatcacttga agtcaggagg 20100 agttcatgac cagcctggcc aacacagcga aaccctgtct ctactaaaaa tacaatacaa 20160 aaattagctg tgcatggtag catgtgcttc tggtcccagc tactcaggcg ctgagacagg 20220 agaagtgcct gaacctggga ggcggaggtt gcagtgagct gagatcaggt cactgcactc 20280 tagcctgggc gacagagcaa gatgcaagac tctgtccccc tccaaaaaaa aaaaaaaaaa 20340 aaaaattaga agaaatcata tgaaaaaaca taaaaatgaa cgacagtgga cttagttctt 20400 ctgggagaac tttgctagct tgaaaattag tgtcaagctg gacaggtggc ttgtgcctgt 20460 aattccagct acttgggagg ctgagacagg aggagcttga gcccaggagc tcaaggctgc 20520 agtgagcctt gattgcacca ctagattcca gcctgggcga cagagcgaga ctctatctct 20580 aaaagaaagg aaaattagta ttataaatta gaaaattaga ggctttgtac aaagctttga 20640 agagttaata ttttaaagta ggagaaaaat gaatcctctg gttatattgc cacaagtaat 20700 tttgcgttga aacactgtgg cccatatagc ctgaagagac tttgaaaggt catttgagcc 20760 cttgagttca gaacctatct cttctgccaa gtgatgacac agaatatgat ctagctcaga 20820 aaagttttac aatctgggtg gtttgtctct ctttttctct ctctcttttt ctttggctga 20880 agaatcttta gaacagtcaa ccttctctta aattgcaagt ttcctttggc catttgaaac 20940 attttctttt tttcttttct ttttcctttt tctttttttg taaagagaca gggcctagct 21000 ctgtcactta gactggaatt tagcggtgtg atcataattc actgtagccg tgaactcctg 21060 gattcaaggg atcttcccat ctcagtctct tgagtagctg ggaatacagg cttgcgccac 21120 cacattcagc taagtttttt atttttttgt agtgctgtgg cctcactgtg tttcccaggt 21180 tggtcttgaa ctcctggcct caagtgacct tcccttgttg gcctcccaaa gcactgggat 21240 tacagacatc agccactgca cccagccaga cattttctat ttacttgtat ggacttaatt 21300 ctgtctatcc cattccccct tcccccatta taattttttt tctctgtaat ttgtaggtac 21360 aagttctttc tctcactgaa aagctttcca attgtagttt ttctgcattg atggaggtag 21420 tgaaggggag gttcaggtaa tttacaacta ttgtaaacag tttttatttg gcatggtaca 21480 gtgtctaatg cctgtaatcc cagcactttg ggaggctgag gcggggaaaa ttgcaagttt 21540 actttgagtt tgagactagc ccggccaaca tcgcgaaacc ctgtctctac caaaaataca 21600 aaaaaattag ccgggtgtgg tggtgcacac ctgaaatccc agctgttctg gaagctgagg 21660 cgtgagaatc gcttgaatcc gggaggtgaa agttgtagtg agctgaggtc acaccactgc 21720 actccagcct gggcgacaga gggagagact ctgtcttaaa aaaagagatt ttatttaagg 21780 gatcatacag agccctaaaa ttatatattc acatttggaa tgttaagcag atgctttgac 21840 atgatatatt ataaatctgt aataataaat agtagttaat cctcatatac tttagtaatt 21900 agcacagtcc aatttttttt ttttttgaga tggagtctca ctctgttgcc caggctggag 21960 tgcagtggta tgatctcggc tcactgcagc ctctgccttc catgttcaag tgattctcct 22020 ggctcagcct cccgagtaac tgggactaca ggtgtgtgcc accacgcctg gctaattttt 22080 tgtattttta gtagagacgg ggtttcactg tcttagccag gatggtctcg atcttctgac 22140 cttgtgatct gcctgtctca gcctcccaaa gtgagccacc gtgcccagcc agcacaatct 22200 tattttttag taggcatgta atttctaaat ttgtctttat tgctaaagta acatcccatt 22260 tatctcaaac taactgtcat cagtcttgtc ttgttctgaa atagcattaa aatatttatc 22320 acaccagcct ctttcctggt catcatgttc tttatgctgt aattattatt gtttttgtta 22380 ttatcatatg ataattttgc cactgcttat tcagtgctta atatatgtca gtcttttctt 22440 acattatatg tagttttttc ttatttgaag acagagtttc ttgctctgtt gcctgggcag 22500 gaatatagta gcgtgatcat ggctgactgt aaccttgacc tcttgggccc aggtgatcct 22560 cccaccttag cctcctgagt agctgggagt acaggtgtcc accaccatgc ctggcgaatt 22620 tttaactttt ttcatagaga cagggtctca ctatgttgcc taggctgatc ttgaactccc 22680 cggttcaagt gatccccctg cctctgcctc ctaaagtgct gggattatag gcgtgagtcc 22740 ccgtgcctgg cccatttaaa attttaattc tcaaaatacc ctgagaggta catagatata 22800 ttcttatttt acagatgaaa aactcaaggc cgagagagat tatataattt acctaactaa 22860 ggtcatgcca ctagtaagtg gcatagtgag gatttaaacc taaactactt caaaccagag 22920 ctcctactaa tattaatgct gtttctgcct ctttaatata tatctttact tggatatcta 22980 gttttcttag tacacagaac atttagaagt gccaaatact gtgtgagttg ttagttgtaa 23040 gtgtatgtgt gtttttatgt gtgtatcgag agggaggagt tttgaacaaa tccggaagtc 23100 agaggtctga cgtgggtctc actgggcatt tcttcttcag gctctagggg agaattggtt 23160 tccttgcctc ttttccagct tctagaggct gtatttcctc ctttgcctct ggtctctcct 23220 attttcaaag ccagcagcct ggtgtcttca aatctgtctc tgacactatc tctcctgcct 23280 ccctctttca cttataagta tccttgtgtt tatattaaat agggctcacc cagataatac 23340 aagctaatct ccccctttca aggtcaacta attagcaact ctgattccat gtcagcttta 23400 aatttcccct tgatagataa cattttcaca tgttttggag attcggatgt ggtcattttg 23460 tggggagggg gagtgcattc tgcctaccag agtttcctta gtgtggcaaa atgatttagc 23520 ttcttaggcc tcgctataaa atgagattag tatttactat ttaccacaaa ggaattttat 23580 gaagatggaa attacgtact aatacagaca ggccatgcat agaatacact taattagcag 23640 atgctttctg cttgatctta tctgctaggt atatgcttgt ggtgatgggt agtctccaag 23700 gtaaccactg ggtttgataa actccaattt gagtgcctga ctcttctaaa ggcagatgtt 23760 tgttttagaa ttcagccttt ccagaccttt gcatgatgag gatggtgatg atttatagca 23820 caatggtgga aaaacagatg agtgcccctt aactcttaat ttgttttatg atttgaatct 23880 gtcttaatct ctaagataca gttcactttt taagtaggca gcttttctgt ttaacatgat 23940 tttctaaagg attgctatag aagataatga aaagggacct tgagtaattg cacttttaaa 24000 tcacacaaga ttgttttgta tgtgctcaga tggtattttt tgaagttaat gttgtcattt 24060 cttttctttt tttttatttt cgggacaggg tcttcctctg ttgcccaggc tggagtgctg 24120 tggtgtgatc ttggctcact gcagcctcca cctcctgggc tcaagtgatc cacccacctc 24180 agcctcccaa gtagctgaga ctacaggtgt atgccaccac gcctggcaag ttttttgtag 24240 aggcagggtt tcaccatgtt gccacgctgg tctcaaactc ctggactcaa gatatctgtc 24300 caccgcagcc ttccaaagtg ctgtgataat aggcgtgagc caccgtgcct ggcctaggca 24360 atttacttct ttgagtttta ttttcagtat atgagaaatg tggctaataa catttacttc 24420 attgcttgct ttgttattaa atatgatgat gcatgtaaaa tactcatgtt tggtttatat 24480 gtgccaaaaa aataatacct gtaatatatt gtagagttag ggatgaacca ctttactaaa 24540 tgtctccaaa taaccaattt ctaataattt agaatatgta gtatagctag tgtgcattgc 24600 attatgataa aagagtgctt attacctgtg cacctaattt ttggaagtca gattcatata 24660 gctttggtta atctttgttc tttttacatt ttattgtatt ttagacaggg tctcgctgtg 24720 ttctgcaggc tgcagtgcaa tggcatgatc atagctcact gcagccttga acccctgggc 24780 tcaagtgatc ctcccacttt agtgtcccaa gtattaaata gctggcatta cagacatgtg 24840 ccaccatgcc tggctgtttc tcgttttttt tagagatggg atctcactat gttgccaagg 24900 ctggtctcga acttctggcc tcaaatgatc ttcttgcctt ggcccctcaa agtgctggat 24960 tacaggagtg agctactgtg tccagcctaa tcttcgttct tggagtcaag ttgtgtaggc 25020 tttgtttttt gctttgcttt tttttttttc ccccacctct agtttttaat ttaaaaaagg 25080 actggctttt agaaccactg gaaaatattt gttttggggg cagtgtcttt agataacata 25140 aaattcagga atacaaattt tgggtggaag atagtacagc gtcattggtt aagaatataa 25200 actctctggc cgggtgtggt ggctcaggcc tgtaatccta gcactctggg aggccgaggc 25260 aggcggatca caaggtcagg agattgagac catcgtggct aacacggtga aaccctgtct 25320 ctactaaaaa tacaaaaaat tagccgggtg tggtggcggg cgcctgtagt cccagctagt 25380 cgagaggctg aggcaggaga atggcgtgaa cccgggaggc ggagcttgca ttgagccgat 25440 cgcgccactg cactccagcc taggcgacag agcgagactc cgtctcaaaa aaaagaaaaa 25500 aagaagaata taaactctca aactgaattc taatcctggc actattactt actgccttgt 25560 gatgttaggc aagtaatgta accttctgtg atgcttaaat tttaatcttg tctgaaatga 25620 ggacaatgat aaaatgtacc tcctactttg gaaggattga atgaggtaat ccatgtaaag 25680 catttagcat ggcctctact aagtgaccca cagcagttat tagtaatgac aaataagaga 25740 aaggagaagc agtgggcagg tgttcaagtg ctaacgtgta ttgtttagag cagtgttatc 25800 aataggagta taatgcaagc catgtgtaat tttaactttt ataatagctg tattaagtaa 25860 ataaaaagaa acaggtgttg aaattcatta tgataatgtt taatttaacc cagcatatcc 25920 aaaacattgt tatttcaaca tgtaatgata tcaaaattat tgagatattt tacatttttt 25980 aatactatat ctttgaaatc tggtgtatat ttatacctat ggcatgtctc aatttggata 26040 ctaaattctt tctgtctttc tgtccccact cctttccctc ccctcccctc ccctcctttc 26100 ccctccttct cttccacttc cctttccctt tttccttcct tccttccttc ctcccctccc 26160 tccctttaca cttccccctc cccatctctt tccccttccc ctttcccttc cccttccctt 26220 ccttttcctt gtttctttgt ttttttttgc tgtttttttt tttttttttt ttttccgttt 26280 tttgagacag agtcagccag gcacagtggc ttatgcttgt aataccagca ctttgggaag 26340 ccgaggtggg tggatcactt gagcctagga gtttgagacc aggttgggca acatggcgaa 26400 accccgtttc cactaaaaat ataaaagagt tagttggctg ggcgcggtgg cccatgcctg 26460 taatcctagc actttgggag gccgaggtgg gcggatcacc tgaggtcagg agttttgaaa 26520 ccccctctct actaaaaaca caaaaattag ctgggtgtgg tagcaggcac ctgtaatccc 26580 agttacttgg gaggctgagg caggagaatc acttgaactt gggaggcaga ggttgcagtg 26640 agctgagatc aggccactgc actccagcct gggtgataag agcgagactg catctcacaa 26700 aaaaaaaaga attagttagg caaggtggtg ggcacctgtg gtcccagcta ctccggtagc 26760 tgaggtggga ggattgcttg agcctgggag gtggagggag gttgcagtga gctgagattg 26820 caccactgca ccctagcctg ggccagggca aggccaccct gtctcaaaag gagaaaaaaa 26880 aaaaaaaaaa aaagaaatag ggtctcactc tcaccggctg gagtacagtg gtgtgatcac 26940 agctcactgc aaccttgact ttctaggctc tagcgatcct cccacctcag tctcccaagt 27000 aattaggact acaagtgtgt gccagcacgc ttggctaatt ttttgtattt tttgtaaaga 27060 caaggtttca ccatgttgcc caggctggtc tcaaactcct gggctcaagc gattctcccg 27120 ccttggcctc ccaaagtgtt cagataacag gcgtgagcca ccacactggg cactaaattt 27180 tcagcagaga tgcttgttct gtatttagat ttaataaaat ttacagtaga aaaagtagat 27240 tcgcctgggt gtggtggctc acgcctataa tcccaacact ttgggaggct gaggcaggaa 27300 gattgtttga gcccaggaat tcaaaaccag cctgggaaac atagtgagac cctgtctgta 27360 tactaaaaaa aaaaaaaaaa aaaaaaaaaa agaatactta aaaaaatgta ttgttggctg 27420 ggcgtagtgg ctcacgccta taatcccagc actctgggag gccgaggcag gcagatcatg 27480 aggtcaagag attgagacca tcctggccaa tatagtgaaa ccccgcctct actaaaaata 27540 tacacacaca aaattagctg gacgtggtgg catgcacctg tagtcccagc tactcgggag 27600 gctgaggcag gagaatcgct tgaacctggg aggtggaggt tgcagtgagc tgagatcgtg 27660 ccactgcact tcagcctggc tacagagcga gactctgtct cacaaaaaat aaaaaaaaaa 27720 gtattcttgt agttcctttt ttctccttgc aaggcatctt taatgtagaa taagctaaat 27780 atagtaataa taataataat taaaaatgat gaagtttatt gagtactgct ttgtatgtgt 27840 tatgttgtct tctaagcaca gatatctcat ttaatcttca tgatagcact atgagaatac 27900 tttaattttc cctgtttaca gatagggaaa atggggacca gagaaagtta tagagcatag 27960 agttgaactc aattttgacc tccagagtct gtttttcttt tttttttttt tatgagacgg 28020 agtctcattc tgtcgcccag gctggagtgc agtggcatga tctcggctca ctacaacctc 28080 cacctcctgg gttcaagtga ttcttgtgcc tcagcctccc aagtagctgg gattgcaggc 28140 acccgccacc ccacctggct aaattttgta ttttttggta gagatggggt tttgccatgt 28200 tggccagggt gttctcggac tcctgacctc aagcggtttg cccgcctcag cctcccaaag 28260 tgctgggatt acaggtatga gccactgtgc ctggcccaga gcctgttttt ctgacagtca 28320 caccgcttat tggttgctgg atagaatgaa aaagcaaatg gaagtacctc tccccaccag 28380 tagtttttct gggtttcttt tatgggggag cgagggtaga gtaacatgtg ggtagaggga 28440 cagtgtttca ggaacccatt tggatattta agaacaggac taaatactta aattacttct 28500 gtgacaataa aaagataccc aggaagctac actctttttt agttctatat tttttgcatc 28560 atacgtttaa aaattggttg ctcctaaatg aagctttaga atcctttgca agatagtact 28620 actgcttcaa gcagctaacc accaatgaac aagaattatg agttcaaact ctggcataga 28680 ccaaccccct aacttggatc tcctaactca taatccagag ttcttttcca gagaactgct 28740 ccgcttttaa aaaccagtgc caggattcag atctcaaatg ataccattaa acactgatag 28800 gcaagaagca tttgtttcta tgagtaaata actctctgat gtattagagg ctggtgttaa 28860 tttttgacta ggctgaactc tgagcgttta ttatttcttc aggtattaaa ccttcagata 28920 atttcaaaaa cacagtttag gccaggcaca gtggctcacg cctttaatct gagtactatg 28980 ggaggcagag gtgggtggat aacaaggtca ggagttcaag aaaccccgtc tctactaaaa 29040 atacaaaaaa tagctgggcg tggtggtggg ctcctgtaat cccagctact cgggcggctg 29100 aggcaggaga atcgtttgaa ccctggaggc ggaggttgca gtgagccaag atcacgccat 29160 tgcactccag cctgggtgac agggcaagac tctgtctcaa taaaaaacaa aaccaaacca 29220 cagtttatcc cttaggaaaa caccaaaaag cttcttatgt acatgaactt gtattagggt 29280 cacactttaa ggtggctcta gaaagtataa tgagagactt acagtaagac tgcttttttt 29340 ttgacgtggc atctcacttt gttgtctagg ctggcttgga actcctgggt tcaagccatc 29400 ctcttgcctc tgcctcccga gtagctttac aggaggatta caggtgtatg ccactgtgct 29460 gggctgagac tgcagttttt aatgaatgaa atgccactgt gctgggctga gactgcagtt 29520 tttaatgaat gaaatgcctc tgtgcatgga ttgaagccag cttttatggc aaacattaac 29580 agtatggttt ggttccccat ctctgtgccc cttgagttat aacttaaatt gttctgattg 29640 tagtattaac tgtgattagg tgttgatgaa agagatatgg agttctaatt aagtaaaatg 29700 aagatctcaa ataaaattga agtagaatga aacgctatat gtaaatacaa ttctgctaaa 29760 caaatattta actgtggata ggcacagtga ctttataatg cagatagtca ttttcatata 29820 tatggatata tataaatata cattatattc atatatatgg atatatataa atatacattg 29880 tattcatata tatggatata tataaatata cgttatattc atatatatgg atatatataa 29940 atatacgtta tattcatata tatggatata tataatgtaa tacggtaatt gatttttaga 30000 ctaaatatat aagttgaaac ataacatttt ctaatttttg gaaaattagt ggtgttaatt 30060 ctggagacta gtaaaaataa atgattagag acgaatacac ttcatggtaa caaacatgct 30120 ggtgatacag ctttaaaact ttatggaaat ttacaacata caaaaataaa gttcactgat 30180 gttttacata ttcatggcct agcatcacca gttgccaata ttttgccatc ttggtattat 30240 ctgtttcact ctgctgcccc tcttctctcc tttctccctc accatgtagc attttaaagc 30300 aaattccaga tatcatttca cccatatgta tttcggtatg tatctctgac acacttgaac 30360 tttcctttca tagtacaact gtcataccat tgtcacaatg aataacatta acattaattc 30420 cttaatggca tctggtagca agaacatgtt cagattagcc tgtctctaaa atgtctttgc 30480 aatttgttcg tttacattag gacccaaata aagtcttcat attgcagttg gaagatacgg 30540 ttctaaatgg tctcttaaat ctccaccctt ttcatgccct ttatttgatg aagaaactgt 30600 tatttaacct gtagaatttc ccacattctg tttggctgat tacatctttg tggtttggtt 30660 taacccctgt aatttcctgt aaactgaaca ttcgttctag agggtatttt tttttggtga 30720 gcattgttta taagaggtgc tgtattcttt ctattagtta ccatcatgag gttggttcac 30780 tttcagtatt gctgggattg gtcagtggtt ttagattatg ttggcttgat ctatccttta 30840 taaagttctt atcaactttc acacaaaggt tttcgcagca tctgaggatt gttgactaga 30900 tccattatat aaggattgca aaacggtgat tttggaatta tatcactcct acatttatta 30960 gttgaaattc ttttgtaaag aaaactttta tttcatgtat ttactgtctt taaaatctag 31020 aagtggaggc tataaaaatt taattcctct cccacctcgt tttgccagtt tttataataa 31080 tgagttggtg tcctaggaac ccactaaaac gaccagtaaa tttttcttct ttcttttatt 31140 atttttagtg ttagggattt ttatgtatgt gatgtgtttt aatccattgt agttgctgtt 31200 tttcatgttt caattgtctc atctttggcc agttggaacc ccttcagatg ggttcctgtg 31260 ttcttttgaa tattgtccca tcggttttca ataacttact ctctttctgg cacaagggat 31320 atcaggttca ctttgtacat ttcctgctcc gcatctggaa tcagctattt ctttaaggaa 31380 ccctggttgc tgagacatta atgacatcaa atatatatat aaaaagaaaa gaacaagtag 31440 ccctggtttt ctcaagtggg aaatggtttt tagagatcac aatcagggtg ttaagggtgc 31500 tcattgctgc tgggttggtc atgacttcta tggtttttta gtatttagat acaggaagta 31560 ctctctccct tccttccctc cctcctctcc ctccctccct ccctctttct ctcccttcct 31620 cccttcctcc cttccttcct tccttccttc cttccttcct tccttccttc cttccttcct 31680 cctttttttt ttttttaatt agagtaaatt atggctgggt gaggtggctc actcctgtaa 31740 cctaagcact ctgggaggct gaggtgggcg gatcacttga gcccaggagt tcgagactag 31800 cctgggcaac atggcaaaac cctgtctcta caaaaagctc aaaaaaatta gccaggcatg 31860 gtggtgtgtg cctatagtcc caactacttg ggaagatcac ccgagcctgg aggtcaaggc 31920 tgcagtgagt tgagattgtg ccactgcacc ctagcctggg tgatagagtg agaccctgtc 31980 tcaaaaaaaa agagaagaat aaattttcaa ttcatactga tatttccagt tttcatgaaa 32040 aattacagag gttttgttta acttatttta tgatttattt atttatttta tggtgaaaaa 32100 tatgtctatt tagtgttagc cagctgggct cactttagat gatcccaatt ttgttggcaa 32160 cgtcatcata gtcaggaacc ggtagaacat gggccttctt tccatcaggc ctgatcaggg 32220 tgttgatatt ggccatgtca gtgccacaga gctttttctt agcctgtttg atctggtatt 32280 tgttggcctt gacatccaca gtgagcacaa gaatgttgtt gactttctgt tcttttcatg 32340 gctgactcag tggtcagggg aaacttgata gcatagtggt caagctggtt tctcctggac 32400 tggaccagtc ttccaaggat atttgggctg cctccagaac agcagcagtg ttttgggctg 32460 tccttaggtg ggtgacgtga agatcttctc ttttttgtgt ggctgtggat acggatacct 32520 ttcagcactg ctgttttggc ctttagagcc tttggtttgg ctttgacgtt gggagagcag 32580 gggcttccct ctttgcctgt gatgccttct tgatgagtac agccatgtaa cttcttttat 32640 agttgtgtct tttttttttt ttgggacgta gtctcgctct gttgtccagg ctggagtgca 32700 gtggcgtgat cttggctcac cgcaacctcc acctcccagg ttcaagcgat tcccctgcct 32760 cagcctcccg agtagctggg attataggca ccctccacca tgcctggcta atttttgtat 32820 ttttagtaga gtcagggttt cactatattg gccaggctga tctcgaactc ctgacctccg 32880 gatccgcctg ccttggcctc ccaaagtgct gggattacag gcgtgagcct ccgtgcctgg 32940 cctttttttt ttttaaattg agatggagtc ttgctttgtc cccccgcagg ctagagtgca 33000 gtggcatgat ctcggctcac tacaacctct gcctcccagg ttcaagcaat tctcctgcct 33060 tagcatccca agtagctggg attacaggca cgtgccacca tgcctggcta atttttgtat 33120 tattattaga gaccaggttt tgccatgttg gccaggccgg tctcgaactc tggacctcaa 33180 gtgatctacc tgccttagcc tcccaaagtg gtgggattac aggcgtgagc cactatgccc 33240 ggcctatatc attttttatc ttatgctaaa aatgttggtt cctaacaaca ttaacattat 33300 attatacata taacacatat tagctttgag ataacaatac cataatgtag tttgagattc 33360 ctttgtctgt ctatttacat ccttaggctg tattccagta gggatgtaag ctcagaatac 33420 tttttaaatg aaaataaaat tttataaatt ttaataaaaa ataatatttt aaataaatat 33480 taatatttaa gatacttgaa ataataattc tctgtgtatt tatgtcagca gttgaaaata 33540 gaacatttac ttcagtttgg ttttggtttc taaggagtgc tgttttttcc ttttcaattt 33600 ttttgatgta aaatatttac atggtttcaa aatgttgagc atatttttaa aaagatatgg 33660 ctgggcacgg tggcttgcgc ctgtaacccc agcaatttgg gaggctgagg cgggcggata 33720 acttgaggtc aggagttcaa gaccagcctg gccaacatgg tgaaaccccg tctctactaa 33780 aaatacaaaa aatcagccgg gtgtggtagc aagcgcctgt aatcccagct gctcaggagg 33840 cttgaacccc tgggttcaag cgggagaatt gcttgaaccc ctgggttcaa gcgggagaat 33900 tgcttgaacc cctgggttca agcgggagaa ttgcttgaac ccgggaggcg gaggttgcag 33960 tgagccgaga tcacacctct gcactccagc ctgggtgaca gagtaagact ctgtctcaaa 34020 aaaaaaaaaa aaaaaatgct gctggtttgc atatcattat tctgcccata tcctatcagt 34080 aacgaaagag gtattatttt actgtatgtt tttacttaaa atttaattta tgcttacatt 34140 aatttttaaa ggttcagatt tgaaagtcca gtgtcaattt tccaggtggc caggtggtac 34200 tagatgtcag taaatagttc ttgtttatat attgcgtttt ataattttaa atattttact 34260 gggttttgga catattgtat gtcaatagaa ggcaactgga actctaattt tagaaactaa 34320 gattatgata tttgtagatg atcagtttta ctccttgtag tccaccccct ccaattactt 34380 aataactatt ttaagatgtt ttacagtcta atatactgaa atttctgaca taaagtcaga 34440 cttccagatt acattgctta aatttgccaa actaggcatt tttctggtgg aggagaaagg 34500 aatcttttcc taaaagtctg gtatatatat attttttgct taatgcctag ttaccatgga 34560 aatagatgtt agaatcagct cttaagtagg taggtagtga caaactggct gtattgctga 34620 attgagttgc tagactgtat ttggcccttt cctgctttca tctttccacc ttctctttca 34680 tcattttttt tttttggttt atgtgctttt tagaatattc tatatagtta aatccaaact 34740 tgacttaacc taggtttgag caacaatttg cttttgctta caatttatac tatctatagg 34800 tagacatttt acctcatatt tacatatgta tagtataagt agataattgt actgacttgg 34860 cacatcagct gttaatgctt ttaagaaaag gtctggaaag acctgtaaca agttaatagt 34920 gatggtctct agctggtgac agtatagaaa atgtttaatg tctactttgt gaagttacaa 34980 tgtttggttt ttaaaatgag catgtactac ttcattatat aaaatttttt tacttgtaga 35040 agtttgtgtt aatgatcagt atttaaaata tagtaaaaaa taagagagga aaagtggaaa 35100 tcctctagta gcatatattt ttttggcctt aactttgacg tttgtgtgat gttgatcttt 35160 agttactgcg cttcggtact gaggatagca gcacctatgt catagtgttg ctgagatgaa 35220 atgagattac taaagcataa tgcttggcac aaggggagca gttaataaaa taggagctct 35280 ggtttttgtg gatcttagat ttgctgccct gtaggttttg ggaaggagta gtctggctct 35340 acaggaatga ggaatgtatt gtggagtctt agaagagcta tctacgtatt catttgaatc 35400 cacaaccttc cctcggagct ttcttcttga ctatgtagtt ctactttagc ttttctcttc 35460 tgtcagtata tttctagtca tttccagttt tctgctcagc agagactttg gggtccatgt 35520 tgtacctgtt ctgttactgt tacataaaag tagatctata aatttatctt gttagtaatc 35580 aaaaaagtta aagttcaagt tttatgcata ttctggatga gttttacctt acccaacctt 35640 taatttggtc atgtgattga aaaaaagatt ttttaggggt tccttttcct atcactgtag 35700 atggaaaatt tggaattttt tttttttttt ttgagacaca gtctcaccct gtccctcagg 35760 ctggagtgca gtggcatgat ctcaactcac tgcagcctct gcctcccggg ttcaagcgtt 35820 cctcctgcct cagcctccca agtagctggg attacaggca tgagccacca caactggcta 35880 cttttgttgt atttttagta gagatggggt ttcaccatgt tggacaggct gttcttgaac 35940 acctgacctc aaatgatcca cctgccttgg cctcccaaag tgctgggatt acaggcgtga 36000 gccactgcac ccagccaaaa cttggaattt ctaagagcct ttattaattt gtaaatcagt 36060 aacattggaa ttgaatattt gaaactggaa ctgtccccag aatattgagg atgcaaagtt 36120 tctgtaggca caattcttca caaaggcaga ctcttgggga caatcaggat gattgcaaaa 36180 tcatatgaaa tataatatgt aacgaaatga agaagcaaac aacttacatt ataagttgta 36240 tctgtattat taaagtccaa atatcctagt ttaatttttt gaatattcaa acatatggct 36300 gaaaagtttt aaaaaatact tataaaagaa tatagtaaaa accctttcat cctacctttt 36360 acctccaatt gttaaatttc ccttctttct gtacagtcat tgtttttgtg tattgtttcc 36420 acacgtagaa aggcaaacac aagtttgccg tcttacttcc ctatctctcc taccctttac 36480 tttcagcttg taaattacac acactgttct gcacttttaa attttgtcac ttaacacatc 36540 tgggagatgt ttgtgttatt attacataga gatcccctca tttgtgggtg tttttttgtt 36600 tgttttgttt ttgttttttt aagacagtct tgctatgtca cccgggctat tttttagtat 36660 tttttgtatt ttttagtaga gacggggttt ggccacgttg gccaggccag tctcaaattc 36720 ctggcctcaa gtgatctgcc cacctcggcc tcccaaagtg ctggaattaa caggtatgag 36780 ccaccacgcc tagccttgtg tgttcttatt aacaactttt attgaggtat aacttgcaca 36840 ccaaaaaaat taaaccattc taagtgtaaa aatcaaggaa ttttagattc atttttaatt 36900 aaaaatttat tttatttatt tatttattta ttattttttt gagacagagt cttgctctgt 36960 tgcccaggct ggagtgcagt ggtgcgatct cagctcaccg caacctctgc ctcccaagtt 37020 cacgccattc tcctgcctca tccttccgag gtagctggga ctacaggcac ccgccaccat 37080 gcccagctaa tttattttta atttttttta tttttagtag agacggggtt tcaacgtgtt 37140 agccagggtg gtcttgatct cctgacctgg tgatctgccc acctccgcat cccaaagtgc 37200 tggaattata ggcgtgagcc accacgcctg gcctattttc ttcttttaga aacaaattgt 37260 tactctgtca cacaggcagg agtgcagtag cactatcata gttcagtgtt aacctcaaac 37320 tcctgggctc aagcaatctt ccaacctcag cctcctgaac aggtgggact gcaggtgtgt 37380 gctaccatgc ccagctaatt aaaacaaatt tgtttgtaca gacacggtag cactatgttg 37440 cctaggctgt tcttgaacac ctcctctcaa gagatactgc cacttggcct cctgaagctc 37500 tgggattaca ggcgtaagcc tccacacctg gctgttatgg aatttttgta aatgtctagg 37560 gttgttcacc catcaccaca gtccagttta agaatatttt tgtaatcctt aaaaagttgt 37620 ctccccgtct ctggtctttc atgcccatgt ccagctgcaa acaaccagtt tcctcaattg 37680 tttttgcagt tctgtatagg acagatgtat aagatttacg atgtaacata atttatttag 37740 ccacacccct atttttggac atttaggttg tttttgacct tttgctttta caaacaggtc 37800 tgagatgagg taactttgta catatattat ttcatacatc tgtaagagta tctgtaggat 37860 taattcctaa gagtagggtt actgattaaa gcacatgtgc tttttttttt tttttggtga 37920 tggaatctca ttctgtcgtc caggttggcg tcatcttggc tcactgcaac ctctgcctcc 37980 ctggttcaag cagttctcct gcctcagcct ctggagtagc tgggattaca ggcatgcacc 38040 accatgcccc gctaagtttt gtatttttag tggagacagg tgtcaccatg ttggccaagt 38100 tggtcttgaa ctcctgacct caagtgatct gcccggcctg cattttttat ttctataatc 38160 gtttcccgtt caggttatac agatttacat tctcagcagc aatatgaagg ctaacatttt 38220 cccataactt acctgccgtg tacattacta gacttctgaa tttctgaatt tttgccaatc 38280 tgatagtgaa aaatggtgtg ggtttttttt ttttagagat agagtctcac tatatcgtcc 38340 aggctggagg gagtgtagtg gcatgatagg ggcacactgc aacctctgtc tcccaggttc 38400 aagcagttct tctgcctcag cctcccaagt agttgaaact atggatgcgt gcgaaaaatt 38460 gtgttttgaa ttgtttttat ttagtaggtt ttacatcgaa tttatgaatg agtatgagca 38520 tcttttcaag tgttgagtcc attgtaattc tttttatatg aaatatcctt ttatgtcata 38580 tgcccatttt tctgttgaat ggttggcttt tttttttttt tttgaggcag aatctccttc 38640 ctgtccccca ggctggagtg cagtggtcca ataacggctt actgcagcct tgaacctccc 38700 aggctcaagt gatcctccca cctcaacctc ctgagtacct ggggctacag gcacatgcta 38760 ccatgctggc taatttttaa attttttaat agagataagg cctcactatg ttgtccaggc 38820 tggtctcgaa ctcctgggct caagcacctc acctacctcg gcctcccagt gttgggacta 38880 taggagtgaa ccactttgcc tggcctgaat ggttggtctc taaaaaaaat tactgatttt 38940 agatgttctt tatgtatgag agtcaagctc tttagtgatg tgaattataa ataacttttt 39000 gtagagtttg tcatttatat tttgatatat ttgctttgtg tatttttttg ccattgagca 39060 attttttaat ggttaaattt atcaatattt tatagctccc aaactttgag ttaatttact 39120 gtttgaagta agtcatacat tgaattccct tccaggatca taatttatta tgtcactttg 39180 aatttagctg gacttttggc cagacctact gacttgcatc tgtgatgaat aactttgaat 39240 atatgttatc tcacacatgt gcaagtatag ctattggatg aatttggcca tttattcctg 39300 gggccagggt tgtgtgtttt taattttggt aattattgtt gaataaatgg atctttcaag 39360 actgttaaga tattgcattg ctaataactg gtgcagaaat taatggtgat ttctgttcaa 39420 gatgccagat tgttaaactc ctctaggatt agaaaagttt tggttgaaaa tagaacactt 39480 aactgtaact aagagtcttc ttttttcatt atgtgttggc ctttaaggca ccagaattat 39540 acttcagaaa ttccttggga gaatttgggt tatgtcttaa aataaaaaat gttcatgttc 39600 tttgtgctcc cttttgatac ctacccagaa gcagcccatg tacaggtgca caaggaagct 39660 ggcatgaata tgtttgtggc tgccctcttt aatagggaaa aacagaaaac cacttaatat 39720 caccagtaga ggacaagcta aataaccatg gcaaattcct acaatggtta tagcacaaaa 39780 ttaattcgac agtttgtgtt tactgatgta gaaagatctc taaaacattt tttgaatgaa 39840 agaaaagttg aaagacagta tattaaatgt agtaccagtt atgtaaaaat agattcacag 39900 aaatgttatt aaatatggat acatgtacgt gtatgtaagt gaaggtctgg aaaaaacctg 39960 gaagaacata tgccaaaatg agaatagtga ttattaatgg gaattgagtg tagtaattta 40020 ggacttactt tttttttttt gaaatggagt ctcactctgt ttttcaggct ggagtgcagt 40080 ggtgcaatct cagcttactg caatctccgt ctcctgagtt caagcagttc tgcctcagcc 40140 tcctgagtag ctgtgactac aggtgcccac caccacgcct ggctaatttt tatatttctt 40200 taatagagaa ggggtttcgc catgttggcc agcctggtct tgagaaattc ctgacttcag 40260 gtgatccacc cgccttggcc tcccaaagtg ctgggattac aggcatgagc cactgcgccc 40320 agccacatta atgttttatt atttttacaa ggataatatg ctcatagact gtacaactaa 40380 aggccaattt aaaatttatt tggaaaaaat acattgtttt gtacataggt ccctgtaaag 40440 gcagagtaag actggaaaaa taccctaaat aaggagacag tagttatgtc cccgagggat 40500 gtgggaacag agaagataaa aggagacatt tgctttttgt atacttctgt atttttatag 40560 ttttataaag caaatgaatt catgcattaa tttgtatgct gaagaaagga ctaaattctt 40620 taggaccagg aattcatgta agagtatctc attttgtcaa gcttatttaa cagtacttag 40680 tgaaaatagt agctgccggt tgaattactg ggtagagaat aaaggtgtca acagaaatag 40740 tttactaatt tgctgataat taggcattaa tttgcttgat gtgatgcatg tttattactt 40800 tagggcaagt agaaaaataa aaataagtag attctttctc atataccatc atgaacactg 40860 gtagtttctg gtacattcaa gacagaacac acaatgagac tgttcttgcc ctgtagcctc 40920 acctggaatt tccttttgtc ttctgctttg ttactacaac ctcccctgca gtttgggtgt 40980 ctctctagtt ctcttttctt tgtttagtag tctgggatca gatctgctct tctgaaatag 41040 aagtcagcag tgaggagaaa aaaggtaaaa aaaaaaaatt tattttttct gagacagagt 41100 cttactctgt cgtccaggct ggagtgcagt ggcacgatct tggctcactc cagcctccgc 41160 ctcttgggtt caagcaattc tcctccctca gcctcctgag tgtctgggac tacaggcacc 41220 tgccaccacg cccggctaat tttgcatttt tagtagagac agggtttcac cgtgttagcc 41280 aggctggtct ctaactcctg acctcaaatg attcacccac ctcaccctcc caaagtgctg 41340 ggattacagg cgtgagccac agtgcctggc caaaaggtaa aatttttaat ccctttattc 41400 agttagtcac taaatgtgag aatgtcttta cccagttctt ctctctccat cccagcatta 41460 gtgcaagcca ttgtatctca ttctagatta tggtagcaga ttcctaactg atctgtctga 41520 ttcagccttg tatttgtaca ttggtatctg tttttcacat tgcagccaca gtgatctaag 41580 acatcagata atgtaactct tgctcataaa actttgtagt agttcttctc ctgttgctat 41640 caggccttgc ttctccagaa ctctggctat ggtcattaat aattgcagct ttctatattg 41700 tctactttcc tcatgcacta tagctcttct ccagaatcta tattgtttct catttcaacc 41760 catcagaatt cttttactag attaccagtc atccttcagg tatccttctc tgagatccta 41820 agatagattt acgtccttct tcgtgctcct cttgtgcaga atctgaccat agtagttaac 41880 ttcatatggt aacttaactg tttaacagat tcctccacta cactccatga aaacaaagac 41940 tgagttttaa atcaagtctc tggttcctaa ccagcacctg atacattata ggctttagtt 42000 taaatgtatg aataagcctg ggtgcgatgg ctcatgcctg taatcccagc actttgggag 42060 gccaaggtgg gaggatcact tgagtccagg agtttgagac cagcctgggt aacatagtga 42120 gaccctgtct ctacaaatta tcaaaaatta gctgggcatt gtggtacatg cctgtagttc 42180 tagctacttg ggaggctgaa acaggctaat tgcttgagcc caggaagtca aggctgcagt 42240 gagccacaga gctcagcttg aataacagag ggagacccta tctcaaaaca aaacaaaaca 42300 aaacccaaaa gccagaaaca aatgtgtgaa tgagcgttaa ctcagtcatt ttttctctca 42360 tatcattttt ttctcttgtt ctttccctac ctagtaatta ataggttgtg gctcagtttg 42420 aaaaacactt gtgtaaggaa tcctgattta ctagttagaa ctctcagaat gagaactctc 42480 tgccagatct ctaaatattt agtaattttt tattctgttg ttaatggtat tttaaaaatt 42540 caatttctga ttgttgctaa tgtataggaa tcctattgac ttctctatat tgtgtatcct 42600 gtgatcttgc taaaaccacc tattggttac agcaggattt ttgtagattc ctttggattt 42660 ccttggtagc ggatcatgtt gtcagtaaag gcagctacta ttcttcatgt ccaattcaaa 42720 tatcttttct ttcttgattg cattggctct ccaaaacaat gcagaataga agtggtgagg 42780 tggatatccc tgtcttgttc ttaattgtag ggaaaagcat tcagtctttc accgttaagt 42840 ataatgttag ttgcagattt ttcatggatg ccttttgtcc gattgaggaa gtttcattct 42900 gttcctagtt tgctgagaga ttacatcagg agtggcttat ggattttgta ttaataaaat 42960 gcattttctg gatatattga ggtaatctgt ttttaaagtt tgttgatacg ttaattacac 43020 tcattgactt ctgaatttta gaacaatcct ccatttctac aataaatgca cttggtcatg 43080 atatattacc ttttagaaca tattgttgga ttctacatgc tagaatttta tttagaattt 43140 ttgggctggg cgtgatggct cacacctcta atcccagcac tttgggttgg cttaagtcca 43200 ggagtctaag actagcctag gcaacatgac aaaaccctgt ctctgcagaa aataaatttt 43260 aaaaaaagtc agctgggttt ggtggtctgc acctgtaggt cccagctact tgggaggctt 43320 gggaggctga ggtgggagga tcacttgact tcaggagttg gaggttgcag tgagccaaga 43380 tggcaccact gaactgcagc ctgggtgata gaacaagacc ctgtctccaa aaaaaaaata 43440 tatatatatt gtttgcattt ttgctcatga gggatattgg tctgtagaga tgtggttttg 43500 ctatgttgcc caggctggtc ttgagctcct ggcctcaagc agtcctctcc tctcagcctc 43560 ccaaagtgtt gggattatag gcatgagcca ccatgtccag cctctagttt tattttctca 43620 taatgtcatt atatatcaga ataatgctgg ctttgtagaa tgagttggga ataattccaa 43680 attttaattt tagagatggt atcttgttat gttgcatagg ctgttctgga actcatgggc 43740 ccaagggatc ctctcacgtt agctttccaa gtagctggga ttatagccat gagccactgt 43800 gcccagcaat ttttatttga aatactaggt cataatcaca ttatggtttt aaagatcact 43860 tgtaatagaa aaagctagtg aagtatatgt aattaaattt ctctgatcaa ttcatcacat 43920 tgcatcatca attttatagg atacttggag agcagataat ttaggccttg aagaattttt 43980 tttcccccag agacggagtc tcgctctgtc gctgaggctg gagtgcagtg gcataatctt 44040 ggctcactgc agcatctgcc tcccaggttc aagcgattct tctgcctcag cctcccgaat 44100 agctgagatt acaggtgcct gccaccacgt ccagctaatt tttgtatgtt tagtagagat 44160 ggggtttcac atgttggcca ggctggtgtc gaactccgac cttcagtgat ctgcctgtct 44220 aggccttcca aagtggtggg attacaggcg tgagccactg tgccggcctg gccttgaaga 44280 attaatgttg atttgtttta aatgtagaat gaggctgggc gtggtggttc agacctgtaa 44340 tcctaacact ttgggaggcc gaggcaggcg gatcacttga ggccgggagt ttgagaccag 44400 tctggccaac atatcgaaac cccgtctcta ctaaaataaa aaaaaaatta gcagggtgtg 44460 gtggcgcatg tctgtaatcc cagctactcg ggaggctgag gcatgagaat cgcttgaact 44520 gggaggcgga ggttgcagtg agccgcgatt gcaccattgc actccagctg gagcaagact 44580 gtcttggaga gcgagactgt ctcaataaat aagtaaataa aggattggtt tcattttctt 44640 tagggtctaa gttaactttt tcttaaaagt atcagcaaga aatcttaaaa ttgactgtct 44700 taagtaaatg ttaaataata gtacttaata ttaataattt tataatttaa taatgaacat 44760 aaagcatttt atttgatttt aacattttct ggttattaaa aaactcatgg tagaaagtat 44820 ggaaaatcca gaaaatcagg taggaaaaag atcactcgta attcagacgt tacagaggta 44880 actactatta acattttagt atatactttc aaatctttac ctgtgtattt cttaaaacat 44940 agtttgtaat catctttttt caacatttta aacattagct tctgaattag taggtaagtt 45000 tatgttggtt acagataggt ttttttaaga gggtgttaag ttgttactac ctgataaggc 45060 agttatggta tgacatctgg atttaagctc tttttcctgc ctctagtgaa ttatgtcctt 45120 aaaatcactt aaagctccac ctctctttgc tcttttatac cagtgggtaa taatactttt 45180 ttatttatct acataattgt ttttgtgatg aaaatgaatg tgttttaaaa gatacataaa 45240 acttcttatt ttgttataaa atgaagtagt atcggccagg tgcggtggct cacgcctgta 45300 atcccagcac tttgggaggc caaggcaggt ggatcacaat aggagtcgag accatcctgg 45360 ctaacacggt gaaaccccgt ctctactaaa aatacaaaaa attagccagg tgtggtggca 45420 ggcgcctgta gtcccaacta ctcgggaggc tgaggcagga gaatggcgtg aacccggtag 45480 gcggaggttg cagtgagccg agatcgtgcc actgcactcc agcctgggtg acagagactc 45540 cgtctcaaaa aaataaataa ataaataaat aaaattaaat agtatgtaat gtaaatattt 45600 ccatgtctgg ttaaatttgg ttgtctttaa gtattcctgt tttctctttc tctaattttt 45660 ctgaaaattc agggtacaat aggagttctt aaactttttt gggtctgtcg aaagtacagg 45720 ctttattgtg tagatatatc atctctgaaa attcttgtag ggtcttcaga aatccacaga 45780 aacagtgtta tggtttccag gttaaaattt tccatattta taatagtttt cacccattgt 45840 ccatggcacc ttggtttacc tacttgtgct tccaaaagtt tgacacactc atacaactaa 45900 ttttcgcatt cactaaattt gcattgactg ttatcccaaa gcctaccaaa aagaaggttg 45960 ctttgcgttg attatatcct gtgaagtagc cttgagcagc aaatattact ttttgtaagt 46020 tagagacagc gctgaggaat aaaatcaatg catttctcaa gtagatcttg ggtttctgag 46080 accatgtaaa aactttgaat cctgaggata taattaaaca aaactgtaaa cagttttcat 46140 aagtatcaca gggctaaggg atgagcaagt gatactaggt taaccaggac tccctgaaga 46200 cattgttaga ttatatgctt taggagtgat tgtgtttttg atgattttca attccttcaa 46260 atatgccact atattctagt atttcactgt ttttatttca tagcagagtc cccttctgaa 46320 tcatggtgta tattcatgat gtatgttcct aggctagtat tttacccaaa ataggaaagt 46380 tttagagatc attttgtatc tgaaaagtat tcccttattt ataatgttga tatctaatat 46440 aagtaatgtg ataatttctt ttaatttttg tatttctgat tttttcagca tttcttattg 46500 gtcttctgaa ttgtataaag aacaggtttt taaaaataca ttcaccagcc tccctaagaa 46560 ttgcatgaga gtacctctta ccactgttaa ataaataact cctgctccct ttgttttttg 46620 tttttgtttt tgtttttctg aggcaggctg ttggtctgtc actcaggctg gagtgcagag 46680 gcatgataac aactcactgc aggcttgatc tcctgggctc aaatgatcct tctacctcag 46740 cctccaccac gagtagctgg ggccacaggc atgggctacc acgctcagct gtttttttga 46800 gacggaatgt cgctctgttg cccaggctgg agtgcagtgg catgatcttg gctcactgca 46860 acctctgcct cttgggttca atcgattctt gtgcctcagc ctcccgtgta gctgggacta 46920 caagcatgca ccaccacgcc cagctgattt ttgcattttt agtagagaca gggtttcacc 46980 acgttggcca ggctagtctt gaactcctta cctcaggtga gccacccgcc tcagcctccc 47040 aaagtgctga gattacaggt atgagccact gtgcccggcc tttatttttg tttttaacag 47100 aggcagtttc cctatgttgc ccaggctggt tttgaactgg gctcaagtga tccacccgcc 47160 ttggcctccc aaagtgctga gattataggc tgagccatcg cacccaaccc tcctgctcac 47220 tttaaagcat gctgtataga gttgtcctgg tgagaagcca ccttgatggc aagcttgtaa 47280 gctgtagagt tggtgagtgt ttacttagga ctcacatttg aaaccgcttt tttttttctg 47340 ttatctttta atatgtaaaa taataatcag aagttactca ggactctttt tttttcgttt 47400 ttatacatat gatttaattt ttgaactgag aattagcgct cacccaatat taagaatttc 47460 tttcaaaaaa cgtgctcggg tcaacacaag gcctctctgt acattatcct tccgaaacca 47520 tggtttcttt gcttgcacct ttatccccac tgccttgcca gtccttcccg ccagcaggac 47580 tggaagcctg gcgactccct aggtcacagg gttttccttg ccataaccaa gtgggctctg 47640 cgcgtggctg atggcagccc caccctgcgc ccctgctgtt aggcaccgag caaggagggg 47700 cccctggctg ttcttcgcct gggcactggt gtgctttcgg cgatcctggc cacctgccca 47760 cctgaagctg ccatcttggg ttctggcagg agccgtttgc gtggcgagga gcggagaggc 47820 aggaacccag tgagctgctc aggacctgag ttgtgggaga tcatgactat ttctttgcga 47880 tgcttcccca atattcttta agtcaatttt gtttgtcaaa tagcctgatt ttagtgatcg 47940 tctcttgtta gagcctgccc agtatgtatt ccaccttgtt tgttaacaac ataccaattt 48000 cccttggatt gtcagtgaag gtgtcctgtt ctttactgta caagaactaa gcatgtgatt 48060 caaataaggt tactggatat gttaaaaaga ataagacaat ggaaataaaa taatattaaa 48120 atgttttatt ttattaatgg gtggagtaag agtaatacaa atgttttaga cttgaagtta 48180 ctgtcagtgt ttttatcatt atatgactgt caaagagctc ataatagaat taagagaatg 48240 tgttggatat ttccagcacc agtgttagat ttggtaattc acaaggtgcg ctcacaggac 48300 tcagtgtatt gtattagtca tgtttacggc tgcgatttat tacaagagga taggaagcat 48360 tatcagcaaa aggaaaagat acattgggta aagtctggag gaaaccaggt gcaaatttct 48420 cccggtagag ttacacaaga cacacataat tcccctaacc aagaattgtg acaacatgaa 48480 atgttacaac ccgggaagct ccttaaaact cagcatccag ggtttttagt gggagctgtt 48540 cacataggta ctgcatgtct ggcatatatc aaattctaga ctctgataat gaaagcttaa 48600 gccatattgt tttagacaca accacccact cttaccagtt ggtggtagga accctcccta 48660 aatccaggtt cctgtgagtt cttagccaag ggccagcttt gcaagcagcc ctttctaagg 48720 aaagccatct caggtctgct gttaactttt ttctgcatag aagattagct ttttatttac 48780 taattcttta gttgtttgcg ttgattattt tatctcaatt gctgatttct ttgctagagt 48840 cctctcaatt tttaaataat gtattatttt aaattggtct gcctttatgg ccgtatttta 48900 ttttgtgtgt atgttacatt gttttcatta tcactatcct gatatctttc aggtctttaa 48960 tgacttctga aatttccaaa atggctttca gattttgatt cctaccagaa tacctcatac 49020 tacgtggttc aggaaactaa atggagattc tgttagtttc tgagcatatc tattacaaat 49080 atctacttaa gacctagagg aataacctct gggtcagtgt ttctcagtct tgccgggggt 49140 ggggggtgca tcagagtgtc ctggagggct tgttaagaca caaaatgctg tttccctccc 49200 ggagagtttc taattcggaa tgtctggagc ggtgcctggg aatttgcatt tctaacaaat 49260 ttccaggtgt tggcttggcg ctgtatgcac tttgctgtaa tcccagcact ttgggaggct 49320 gaggtgggtg gaccacttca gcctgggagg tggaggctgc agtgagttag gatttccagc 49380 ctgggtgaca gagtgagtga gaccctgtct caaaaaaaca aattttcaga tgttgctgat 49440 ggtgttggtc ctgggagcat actttgggaa ccacttttct agtttataat acaatccaca 49500 aacttatgta aacaagaaaa atgtaatctg taaaattccc atgtacagga ataccaaata 49560 cagtgtgcca aaagtggtag gacaattgaa gacttcattg tcaaccctgc tgtgttgtgc 49620 agctgccact tttccctcag aatgtgtgtg ttcacaaggt gtgttgcttt tgtagatcaa 49680 cataagaaat acttactagt aaagtttatt tcttatctgt ttttaagata agtaaattaa 49740 cagttcacta ttttttgtgt gtgctcagtt aatcttcctc atatcacctg gaatttatca 49800 tctcccttgg gtaatcacag tctagtagat tctctcttca ccattttctt agggaaaatg 49860 gattggcaag ggaaatgcta gactatttta agggatttat tggcttttct gtcccactgg 49920 gaatgcttat aggaggggat gccatgaaat gtttttcatg atctcagtgg ggatcagaaa 49980 attccagcag aacggaggct aggtgggcat ggttaacaca tttaggtagg agggcatgtt 50040 tgataatgaa attctgaaaa tgtttgcatg tgcattgaga tccctaggta gataagtaac 50100 ctagtaactt atttgattgg tatagttttt taaaactcca ggtctgatga taatctaata 50160 taaactattc tattgaatgc tttgaaattg agtctgttta gttctgagag gccattgaaa 50220 gaaggcaaga ccgagtatcc tggaaggcct gttacatacc cctgtatgta gcatggccct 50280 ttctacttgc ctttgctaat gatactttgt gtgtgtacat ttacttaaat agctgtgcag 50340 tgggataggg gaataatact caggttttaa aatctgattt tggctctgac ttaaccatta 50400 attactggtg actcaaaaga ataccactgt gacctaccag taagagtagg tgcatatgga 50460 gagtcaggaa atagcatttt gatagagttt tgacttaaat atgtttcctt acactcatct 50520 tgaaattgtt tcttatggtc ctaagtgaat agttggaata gatatcctca gcaactaata 50580 gaatctccat ttagtgtgag gtagtatgga aggaatggcc aagaaaagcc atttagtgcc 50640 accattaaaa tcctgaaaga tgctgggcaa ggtcatcagg agaaaaatgt aaaacaaaac 50700 caatgaaaca cacacacaca cacacacaca cacacacaca cacacacaca caatataagg 50760 cattaaaaat ggtctcattt acattattat tattattttt tttttttttt gagacggagt 50820 ctcgctgtcg cccaggctgg agtgcagtgg cgggatctcg gctcactgca agctccgcct 50880 cccgggttca cgccattctc ctgcctcagc ctcccgagta gctgggatta caggcgtgcg 50940 ccaccatgcc aggctaattt tgtattttta gtagagaccg ggtttcacta tgttggtcag 51000 gctggtcttg aacttctgac ctcaggtgat ctacccttct cagcctccca tagtgctggg 51060 attacactgc acccagcctc taaaatcttt tttaaatttt agagagagag tctctctctg 51120 ccacccaaac tgaagtgcag tggcacgatc atgtcttgtt gcacctttgg cctcctgagc 51180 tcaagtgatc ttctcacctc agccccctga atggttggga ccataggcgt gtgccactga 51240 gcccagataa tttttttatt ttttatagag ccatgtcttc ctttgttgct cagggttttt 51300 cttggcaaaa gcatattgcc agaacatatc tttttttttt tttttcggag atggtgtttt 51360 gctccatcac ctaggcttga ttgcagtgca caatctcgac tcactgcaat ctccacctcc 51420 cgggttcaag cgattctcct gcctcagcct cccgagtagc tgggattaca ggcacccgct 51480 accactccca gctaattttt tgtattttta gtagagatga ggtttcacca tgttggccag 51540 gctggtctca aactcctgac ttcaggtgat cccccctgct tcggcctccc aaagtgctgg 51600 gattataggc atgagccacc gcgcctggcc aacatctctt ttttaagaga aataacttgt 51660 aatttctttc tttccttttt taaagagaca gggtcttgct gtgtctccca gactggagtg 51720 cagtggtgtg atcatagctc aatacagcct tgaacttctg ggctcaagca atcctactgc 51780 ctcagcctca caagtagcta ggactacaga tgtgcaccac catgacaatt agtattcttg 51840 ctttattttt tctttttatt tgataagaag tttataactt tgaaaaatat gagtagaaat 51900 gtaagagtct gttgaagatg taaactattg ttttttaaaa agtgtaaata tatttttaat 51960 aagattgtag ggccagacgc atctggtaag ggcttaaaag ctgattttta aaaattgata 52020 gccagaggtt atatcttact aatgtttgct aatgctacta attagcgtta gtttgctaat 52080 gctactaatg ttataataat gatctgatga cctcaaatag cttttcaaaa aaaaaatctt 52140 tttttttttt tttttgcttc gtatgttcac atttcattat ttcttactgg tcaagatttt 52200 ttttcttttt ttgaaagtct ctgtctgtca cctaggctgg agtgcaatgg tgtgatcaca 52260 gcttgctgca gcctctactc cccaggctca agtgatcctt ctgcctcagc cccctcacta 52320 gctaggagcc acaggtgtgt gctaccacgc ctggctaagt ttttgatatt ttgtagagat 52380 gaggttttgc cgtgttgtcc cggctggtct tgaactcctg ggctcaagca gtcctcccac 52440 cttggccttc cagcgtgttg ggattacagg cgtgagccac tgtgcccagc ccaggatttt 52500 tgtatatttt gacagtttag cttaaaaatg taaactggaa ttggtttaat atttgtctag 52560 atgtgatcta agttctgaat atcccagggc attgtgggtg ctcaattaat gtttgtggaa 52620 ttgtaactgt atttgatatt atttctattt tgataatagg catctgatgg gtttaattaa 52680 agctattttg gttatgggta atagaaccca tttcaggtga tgaaaagcag aaaagagaat 52740 ctattataag ggtacagagc atctcatggt acccaaagag gggaactcca gtttggcttg 52800 aggaggcagt tggaaagcta gcaggaacta aggctagcta ttctctcagt cactcagaga 52860 acccacagtt tcttatgtgc actttttccc cctctgcatc tgttttattt cttacttcct 52920 ctcagcagac tggttttctc tacttctccc tgtgcatgcc agaaaatggc taccataggc 52980 tgcatcacat cctttctcta ccggctaatt ggttaaccta gctttaactt catagcagag 53040 taaaaccgat cggcccagtt tggatcaggt ggctagcagt ctaatgaatt acttgttggc 53100 aaagcatatt gccagaacat atctctttgt taagagaaat aacttgtaat tgatacttgt 53160 catgttagtg acttagtctt tgtaaagctg tgtgggcttc tttgtcgtaa aaatttggtc 53220 atctgtaaga tagtttgtcc ttattttact tattaaaata tgatttgttg ggtccctcat 53280 atgtgcatag tcattgaata ggcagagcgg tctgcgcgcg tgcacacaca cacacacaca 53340 cacacacaca caaaattagg cctagtgttg gattttccta ctcttaccat tgtgcctgtt 53400 tttagtatgc atttgattta gcacatgata atcataattc aaagataagt ctgcagcaga 53460 cttaacattt atatataaag tgtttttaag gcagaaggta ttatttaccc tctttttaga 53520 agtggagaaa ctgagaatca gggagtaaaa atcagtcaac taataggttg cagatcgtag 53580 ccatagctga gttgagatat tctttacaca aattatggcc ttattcttct atatcctttt 53640 acctctatgt gagtgtatat caaactggac ttctccgaaa ttctctttgg cctagtttgc 53700 aagttctctg tgtagaataa aaaattacat ttttaattga ttatttttat caagtaatac 53760 atgcaaatga cactaaattc aaaaggtata tactttatta ttgttttatc ttgagtttct 53820 tccatttcat ttcatacaga gagctaaaaa gatttaatat aaacattttc tgggtcattt 53880 gtacatctgg atagcagaga actattaggt tggtgcaaaa gtaattgtgg ttttgcaatt 53940 acttttattt ttatttattt atttattatt tttgagtcgg agtctcactg tcacccaggc 54000 tggagtgcag tggtgtaatc tcagctcact gcaacctcca cctcccgggc tcaagtgatt 54060 ctctctgcct caacctccca agtagcctcc caagccacca ccacgcccag ctaatttttt 54120 ttttttgtat tttagtagct tttttgtatt tcaccatgtt gcccagagtg gtcttgaact 54180 cttgagctca ggtgatccac ctgccttggc ctcccaaagt gctgggatta caggtgtgag 54240 ccactgtgcc tggcatgcaa ttacttttaa tggcaaagac cacaattagt tttgcaccaa 54300 actagtataa tttcagtttc tgcaattact ttttatgttt tacaaccagc gttcatagtg 54360 agtagtatta cttcgcttgt tgcaggtttg attataaaac aacggagatg gagttcacag 54420 taagtgatat gttcctacat catgaggcaa cattgatctc tccagcaaag gttttgtact 54480 agtttacata gggaaaagtg gtgggagaat tgattcgtta cactgtgctc aatattaaaa 54540 gcatgctagt ccaaagtagt ctgtgccata tttgtgtttt gagtgtgtag ttgtagcaga 54600 atatcatcca tcatattgaa ttaatgttct tacttgaatt tcattgattt tgttggtttg 54660 tggaatttag tttgcagatt tggttttata cttgtacagt tccgtaagaa taaataattc 54720 ttagctacta tatgactaca ttttatcagg tacaagtata cattttctac attttaacat 54780 ctctgaaatc aggatgcatt tagcagtcaa ttgatggtat atcgtagttt aattggcagc 54840 ttttttcttt tttagtggta ggtgtggttt acaattagtg acttaaatta gatgaaatat 54900 ggtaacttta tgtttgtatg aatttcagta acataaaaat aaaccatcat ctctgtaaag 54960 gagagataat ttactttttt taagtttaag aacagctgtg taggctgagt gcggtggctc 55020 atgcttgtag tcctagcact ttgggaggcc aaggctggcg gatcatctga ggtcaggagt 55080 tcgagaccag cctggccaac atggtgaaaa ctcgtctcta taaaaataca cacacatgaa 55140 attagctggg tggggtggtg cacgcctgta gtcccagcta cttggaaggc tgaggcagga 55200 gaatcacttg aacctgggag gcagaggttg cagtgagcca agattgcgcc actgcactct 55260 agcctgtaaa tataactgcc aggaacttga gatttcagaa aacatttctc cagagtatct 55320 ggaaaatcac ctggcttacc caaacaaaat tgccacttgt ggatatttgc taccaatgcc 55380 cctatcattc ttctctgtag tcctgtactc ataagcttag ctgcttagct gctcatacct 55440 attcttgaac tgcttctgtc actaatttct acgtgatctg attgttaagt acaaaatctt 55500 ctttaaaaag aatggtaata ataaattaat atagacattt atttatattg agtgcttatt 55560 acatgtgatt ttatgtgtaa taggccacat tggtattgta tgtatttaat atgtaattta 55620 tatttaatta taatatgtaa taaatgtata atataaaagt gaacatatgt tttattagta 55680 tgataaacta cactttacaa atgtgaagct atgctcagat aagttaaata atttttccca 55740 aggatgatgg atctagtaag ttatgaacct aaagtttgat ctcagatatt gattccatag 55800 ctcatgcttt ttcacctgtt tatataagac gcttctgatt ataaggtatt tttgaaatat 55860 aaataatatt caacttaaga ggaatctcat ttagcagggc ttaactaatg tctaatcttt 55920 tttgtgttaa caattatctc atgaatttct tctatatgta ggtgctattc taggaccagg 55980 gcattcatag gtgaataaga cactatttct atcttaagta gctcagtcta gaggacagat 56040 aggaatcatt aaaatatggt ataaaatggt cacttttgca aaatgttggt atatttggga 56100 aaaggaaagt cagtatatag tgttacatca atgagtattt agcataagtt accaaagggc 56160 ttactacagt aatattctga atctagaatg aattttgtga cttagtgttt aatacatgtt 56220 gtgtgggtcc gtttcttttg gtcactagac aagtaattga taggttttca agtgattaaa 56280 gattgatgtt tcttgttgat gacgtctttc aaaatcatat tcttgaggat atgaaacacg 56340 attaaaaact tggttggtgg ggattattat tattatttat tattattatt actttttgag 56400 atggagtctc gctctgttgc ccaggttgga gtgcagtggc gtgatctcgg ctcactgcaa 56460 cctccacctc ctgggtttaa gtgattctcg tgactcaacc cgtgagtagc tggcattaca 56520 ggtgcctgcc accatggcca gctaattttt tttattttta ttagagacag ggtttcacta 56580 tgttggtcag gctggtctca aactccttac ctcaggtgat ctgcccacct tggcctccca 56640 aagtgttggg attacaggtg tgagccaccg tgcctggctg gattattatt attatttatt 56700 ttattttatt tttttgaaac agagtttcac ttttgttgcc caggctggag tgcagtggtg 56760 caaactcggc tcactgcaat ctccgcctcc cgagtccaag tgatactcct gcctcagcct 56820 cccaagtagc tgggattaca ggtgcctacc accatgcctg gctaattttt gtatttttag 56880 tagagactgg gtttcaccat gttggccagg ctggactcga actcctgact gcagatgatc 56940 cgcctgtctc agcctcccaa agtgttgtga ttacaggtgt gagccatggc atccgaccta 57000 tcattttttt agcttggtca tccaggctgg agtgcagtga tatagtcact gcggccacaa 57060 cctcctgggc tcaagcagtt ctcccacctc agcctcctta gtagctggga ccataagtgt 57120 tcaccacctc ttctggctta tttttcaaaa ttatttgtag agatgaagtc tcgctatgtt 57180 gcccaggctc gtctcaaact cctgggctca agtgatcctc ctgccttggt ctcccgaagt 57240 gctgggatta caggactggc ccagtaggga ttttaaggat tttttttttt aaatcaaact 57300 cttttaaatg ttctccagat gctgtcttta gtgacttgtt atactaaaaa atgttctact 57360 tattgccttc taatccatgc cagtagttat tactaacatg cccagataca ttaaaccata 57420 acaatgccag tttctgtttc tgtttgtatt ctgaattttg aactgcctga atcctccact 57480 aggctcctct aatttccaga tcatgaaagt ttatgttctg agagtgctgt tactccaaag 57540 aagattcatt tgcatttgaa tatgattgtg acctcactag caagatgaca aataacctct 57600 tctcaaggca gagtagattg gctgtgttac atgagaaagc tcctttgctt ttttgatact 57660 tagaacagtg ccttaagtat agttggcttt taataatgtc tctcccaatt tctctcttgc 57720 ctgtttgccg aggcagaaaa ttctagttag aatatttctt tggagttact taaatttcca 57780 ggaatatgca gatactcttc tgttaatatt tgtgactatg cagatatccc tctggtttat 57840 gagatgtgga tctaaaattt acttataacc taaagtagct taggttttgt ctcctaaagt 57900 agcttaaatt ttaagaagat acacagtggg gccatgtaaa aaaccaaaaa taaactttaa 57960 aaaattgtaa gaagaatatt aagaaatata gataacatcc aaagattttc gtgttttggg 58020 aaggggttga gtttttttgt tttgttttgt tttttgtttt tttgagacgg agtctctctc 58080 tgtcgcccgg gctggagtcc agtggcatga tctcggctca ctgcaagcac cacctcctgg 58140 gttcacacca ttctcctgcc tcagcctcct gagtggctgg gactacaggc gcctgtcatg 58200 acgcccggct aattttttgt atgtttagta gagacggggt ttcaccgtgt tagccaagat 58260 ggtctcgatc tcctgacctt gtgatctgtc cgcctcggcc tcccaaaatg cttggattac 58320 aggcgtgagg caccgcgccc ggctggcgtt gagttttaaa atatgcagta actcttacaa 58380 tccagcaaaa aaaacagaca aacaaaaaac aaaaacaaaa atgagcaatt cagaaagatg 58440 ggtaaataat ataaatagat aattaataga aaaatatgca aatgcccaat aaatatatat 58500 tactttacaa aataatttat tttcaaataa ttccaaattt gctgtataaa tagttcatgt 58560 aatctccttc accagactgg ccagttgtta acattcgatg tcatttgctc tatatctaat 58620 ttatcttttt ctctgtgtgt gtgtatacac agagagagat atatacacag atttgtatgt 58680 gtgtgtatat atatgtgtgt atatatacac atatatattg tatacatata tgtgtacata 58740 tgtatgtact cattatcatt acgttcaaac tttttaagag caagcttaaa acacaatgtt 58800 ctgtcaccca ttgtactcca gtgtgtaatt cctgataaat agaacagaat aacttcaact 58860 tcggtctgtc tactttttct catttccagt tactcttggc agatatacca cagaaagttg 58920 ctcttttgat actacagtgc aaaacgttgc acaattacac cactaaaaat aattcagtag 58980 tattcaaaga tgatgtgaat attctgttca tcaaaccacc tgtcagctta gcatcttttg 59040 atagctctct gaatcagcca cctttatgat ggttgccaaa cgctgattct ctgtcagttc 59100 tgttacagtt attaacattt tagtataagg aaaagcttta tcttttctac attttaaaac 59160 attcattcat tcattcttgt caatatggac tcacggattt catttttact gaatgcattg 59220 taatttgtta ccaacttttt aaagtgagat ataattaatt tactgtggaa tacacagatc 59280 ttaagttgat cagtttgaca tacatatata tccctaagga gataatagca caaataatat 59340 ttattttaat gctcaaactt tcccccattt gaccagtaag aggaaaaccc cttcaagatg 59400 cttcctgtgt ctgtttgaaa tgtccctatc attaatcgca ccatttcttt actttctgac 59460 acaagatgtt tcagattctt cttgtgcttt tctctgtcca accttgaaat caaccatttt 59520 tctaaggatc ctaattcctt ataatggaga gagttattta gaaatcaaga cgtgggccga 59580 ggcgggtgga tcacgaagtc gggagtttga gaccagcctg accaacatgg tgaaatcccg 59640 tctctactaa ttagctggac atggtggcga gcgcctgtaa tcccagctac ttgggaggct 59700 ggggcaggag aattgcttga acctgggagg cagaggttac agtgagccga tatcacacca 59760 ttgcactcca gcctgggcaa cagagcaaga ctccatctca attaaaaata aataaataaa 59820 tcaagatgta ggctattgct gtttgttgtt gttgttactg ggctgtcttt tggttgctct 59880 tagtactttc tttttggaca gagctaggaa aatataggga cacacacgca cacctagaag 59940 tacttgttta tttttctgtg tgtgtgtgtg tgtgtatgta tatatacaca tatatatata 60000 ttatacaatc atgaattcaa accaaatttc caattccagt ccaatattta gacttctctg 60060 tagtctgctc ccttccttcc attttaactt ctttggagtg aaaaacatgg cccccactat 60120 attcagtgta tttacttatt tgattagtcc atttacttat ttggtccggg taatggatct 60180 tccagccttg cagcttatct cctctgtccc tattcagctc tcatccatgt cctccgccac 60240 aggactgcac ctccatgtgg ttcccccagg cctcctcttc actgccttgt atattcagct 60300 tctgtccttg tgcctattca acccttaccc cttcacaaat ccatgtcctt agtgccacat 60360 gaaaaaggag agaagaaatg gccccagaga atattttgaa gttatattgc atggttttct 60420 tttcttttct tttctttttt tttttgagac gtagtctcac gtcactcagg ttggagtgca 60480 gtgactcgat ctcggctcac tgcaactccg cctcccaggt tcatgccatt ctcctgcctc 60540 agcctcccga gtagctgggc ctacaggtgc ccaccaccat gcccggctac ttttttgtat 60600 ttttagtaga gacggggttt caccttgtta gcctacaggc acccgccacc acacccggct 60660 aattttttat atttttagta gagacggggt ttcaccgggt tagccaggat ggtctccatc 60720 tcctgacctc gtgatccacc cgcctcggcc tcccagagtg ctgggattac aggcgtgagc 60780 caccacacct ggccatggtt ttcttcattt cctcgatgta ttcatttttt ttatattccc 60840 tcccaccaaa ttgtgcattt taagcttggt ttttaaatgg tacactgttt ggtatataaa 60900 tgcaagtata ttttgttcat tcatcttttg gaattatctc cattatttta atagttcatt 60960 gggattgcta tatataaaat aggctgtttt catagattta ttgtcacgtg gtttgtgttt 61020 aagtacattt attaatgatg gttttttgtt ttgttttgtt ttgttttttg acagagcctt 61080 gctctgtcaa cccggctgga gtgccgtgtt gcagtcatgg ctcactgcag cctccatttc 61140 ctgagctcta gcgatcctcc cgcctcagcc tccttagtag ttgggaccat acgtgcatgc 61200 cactatgcct ggctaatttt tgtatttctg gtagagacgg ggtttcaccc tgttacccaa 61260 gctggtcttg aactcctgag ctcaaacaat ctacacacct cgggctccca aagtgctggg 61320 attgtaggtg tgagccattg cacccagcct actgatggtt tttatgatca aatttaatac 61380 tcttgttttc tgattcttct ggctacattg gcactgtaac caatttgaat ccctcacttt 61440 ttaaaataaa ctacctcaat aaaatgtcat ctagcatgtt ttgcttttta aaaaccatct 61500 cgaataattg agtatttcct agtcagtgtc tgtgttagcc agccatttga agatttgatt 61560 taactattgt ctttctaaaa cttaatttat gtatttgatg tgctgcagcc cagagacaca 61620 ttagagatta ctgttgattc atttgtgcac ttctgtgctt ccttggcatt ttggcatctt 61680 gaaggattat tatccactaa tagctaattt taaaattgct tatgcttttt agtgttccca 61740 tggaattcag tgttttttag gtgccactta ggacatttcg tagactggct aggaaaataa 61800 ttatttaagc aatgctggaa aactgtgagg tagctgttgc cttgacaacc agaaaactgt 61860 tctgtttgct caacaaaggg atggtaattt agtagtttaa tttccttttt ggcatggagg 61920 tgctacataa aaacatctta ttttagcctc tgagatgatc catactactg atcagaatta 61980 gagaagcaga gcaaaaagaa aaggcaagag tttttttttc tggctacgta tagatagaca 62040 tgcatattta tcagggttgg tgttggtcaa aagaatctta gttaacctgc tgacataatc 62100 ttttttatgt ctctaaattg gaccaaagta aattaacctc aaaatactgg tgcaatcttg 62160 gtacactaag gggtcgtgat acacttttat tatggagcat ccccacaaag atttaagatt 62220 ctcttgccgc tggaattcaa catgattact tcatgtctgg attgaataag ggagaaaata 62280 ttttaaagga acccctcttc cccacgtata tacacaatta cattggcatc tgctctattt 62340 atgtgggaat tttttttatg acttaaccca tttgtatata gacttaaaca ttttttttta 62400 aaaaacaatt gcctcccagg ttgtaaagta aaaataatag cctttatttg gcaatgtgtg 62460 ctgggccctc ttctaagcat tttgtgtgca ccgtcttact cctcatcctt gtgacagaac 62520 tgttattgta tgctgaggca cttagctaag tggcttagcc caaggtcacc tcactactaa 62580 gggatagatt tgaagctagg catctagttt aggaacatat accctttttt ttcttttctt 62640 ttttttttga gacaaggtct cagtgtcgcc caggttagag tgcggtggcg cgatctctgc 62700 tcactgcaac ctcagtctct caggctgaag tggtcctccc accttagcct ccctcatagc 62760 caggactaca agcaggtgcc accatgcttg gctaattttt gtatttttag tagaaacagg 62820 gttttgccat gttgcccagt ctggttttga actcctgggc tcaagcagtc cgcccactta 62880 ggcttcctaa agtgctggga ttacaggtgt gagccactgt gcttggcttg ggaacctata 62940 ctcttaatta ctgtattata ctgacttttt tttttttttg aaatggagtc tcacagtgtc 63000 gcctgggctg gagtgcaatg gcacaatctt gactcactgc aacctctacc tcccaggttc 63060 aagtgattct cctgctttag cctcccgagt agctgggatt acaggcgccc gccaccacac 63120 ctggctaatg ttttgtattt ttagtagaga gggggttttc accatgttgg ccaggctggt 63180 ctcgaatgcc tgacctcgtg atccacctgc ctcagcctcc caaagtgctg ggattattat 63240 aggcatgagc caccgtgccc agccgaaaac tttttttttt ttttgagacg aagtttcact 63300 cttgttgccc aggctggagt gcaacggcat gatctcggct cactgcaacc tccgcctcct 63360 gagatcaagc gattcttctg cctcagcctc ccgtgtagct gggattacag gcgcccgcca 63420 ccatgcctgg ctaatttttt atattttaag tagagatggg gtttcaccat gttgaccagg 63480 cttgtctcga actcctgacc ttcaggtaat ccacccgcct tggcctccca aagtgctggg 63540 attacaggca tgagccacct tgcccagcca aaatgtggtt ttgcccctcg aatattaaga 63600 agaaataagg aagggaacca aatctgaatt actattgata aaatgacttg tttgtccaca 63660 tcaagtgttt cataatatta attataactt cctagctgac tacctcagac atcacaatgg 63720 agtgctttct aatattgact ctgttttttt gatgtggcag ctgaagctta gagaggtgag 63780 aggttcagta gcctacaaaa actcatctag ttggtgtaag gagtagagct tggattagga 63840 ccttgcctct ctgctttcaa agcctgtgct attaatcagt ctgctctatt acctcatgtt 63900 aaagtaatga tagagtgata ccttatgccc agctaaaatt acatactcaa atctgaccac 63960 ctcagttatg gatttgattt gggtttctgt gtaaagtctc attcatattt tggttggcat 64020 tgaaaaatag tttgacagtt tcattctaga tgattatata tgtctcaaaa cttgccgttc 64080 tgaccacctt tgtaatgcca gtgctttgat ataggcttgc taaacaatta tgtgtaagat 64140 tcaaattatt gtgctgaatg agaatttagg aacagaaaag taacttacct gagataatgt 64200 ataataaata gaggtggcag cagtaagatc agaacacaga tcactgcttt ttctgtgctc 64260 tttctaacga attaacactg ctctgatgat gtgtttcagt tttagcagct tttattagac 64320 tttggtttcc ttctgcaagt cttttttttt tgagatggag tctcgctctg tcacccaggc 64380 tggagtgcag tggcacgatc ttggctcact gcaagccccg cctcccaggt tcacgccatt 64440 ctcctgcctc agcctcctga gtagctagga ctacaggtgt gcgccaccac gcccagctat 64500 ttttttgtat tttttttttt tagtagagac ggggtttcac cgtgttagcc aggatggttt 64560 tgatctcctg acctcgtgat ccgcccgcct cagcctccca aagtggtggg attacaggtg 64620 tgagtcccgc gcccagccct ccttctgcaa gtctttctaa aggcattttc tttatatgcc 64680 ttgtaattcc ttttcctgtg ttctagcatg aaggaaagaa aatattgccc ttcaaaagaa 64740 tagtttgccc cacaataatt tgaagtaata aatacccttc tcccgtcaac acggattttt 64800 atattgttga agatgtggga tgggcttaat ttgggggcgg agggagtccc ctttctcaaa 64860 ttcagcttta ataaatatgc ccaataagca aatctgcagt ttgctgtagt tgaaatgttg 64920 ctagtgtctg tgaatgttaa tgaaaagaat acgcaaatgg gtttctgaat actaatagtc 64980 taaagatgtt agtattctat accctatttt tgttaagaat tatctattaa aaatttaaaa 65040 gtgtatcaat tcctaaattt ttatatgttc ctcagtaggt acaaatatgc aacttttagt 65100 tgatttattg ttctcttcta tgtttcataa ttttagttgc ctcatcagtt ttaatttatt 65160 tttaactata ctgtttgtct ctgaaaataa aattttactg gctagtatgg cagaatgtgt 65220 taatagagga ggttgcaaat tgtggaaagt tatgagcctg tggtcatgaa gactgccctg 65280 ccatttgggt ttttatagca gtaataccca gtaccaattg aaccacaaag tgaactgaaa 65340 tttccttaaa ttgtttctct ctctataaaa catttaaaaa aaatttaatt gatgagtaaa 65400 gcttgaatat attcagtatg tgcaacatga tgaattgatg tacattttat gattaccaca 65460 gttaaattaa caaacacatt cattatcact tgtgctgtac attatattcc catccttatg 65520 ataaaaattt ttttttaatt aattgagata ggatcttgct gtgttgcttg ggctgatctt 65580 gaactcctga gctcaagcag tcctgcttcg gcctcccaaa ttgctgggat tacaggtgtg 65640 agccacaatg cgtggccatt tttatgattt tggagcgaag tggtgtgagt cccttttcat 65700 attattgctt aaataaagcc actagtatgc ctggaatgta accataattt tggccaggga 65760 aaatgatatt ttaagagacc taaggcaggt aggtaaagta gaaatgcatt tattcactag 65820 gtacaacagc aatgtaaagt tatcaagttg tagttaataa tataaaaaaa ttagaaagta 65880 ttagtgcaga aatgattatc tttctgtaag ggtatattcc cattatggta taaatgaact 65940 gatgaatagg cattttccta agttgatttt aaaaattgta taactgatat gttttgagtt 66000 tactttttat gaaatacatg gaatgtgaag caggtggcca ttatggaggt ctgatataat 66060 tgtgggcact cctgttatag cactagaata ttgttttttt ccctttttct ttgggaaata 66120 gattactctg tatgatagct acaactttta ggggagaatt tattttaaaa tctaaatgaa 66180 attattcttt cattatttat tactcgtaac agcatttctc actttatatt ctatgggatt 66240 tctgtaggat gttaatagat gtcaatagaa aagaaagggt tctttggaca aatagattgg 66300 gaaatgttga gttaaaatgt aatcattttc tctattatag ggtttctcag atcttttagc 66360 ttacatttat gaatctccaa agttgaggca gagaagtaat tcattcaata tagtgaattt 66420 cccccaactc ctgcctcctt tccttttggt ggaacgtcct gggatgttag tactttttat 66480 aaagcacttt ggaaaaagct tacttccatg attttccttt ggctatggaa ttactataag 66540 cagaagaact ccttagaaat aaaaatgtag aaatgtacct gaatgtaaaa cgtaagagca 66600 gctattaaaa actaaatcac aactgaggaa acataaatat tagttaaggt agaagagtaa 66660 atatccagcg aatccctcct attaaagcat ttaagatatt ttggcttcaa agttttgaga 66720 ctttccagat gactttcttc atatagtttt cactagtagt ttaaatatac taacttttct 66780 cccttgtatg ctatactaca ttcttttttg tttgcttttt tgagacaggg tcttactctg 66840 tggcccaggc tagagttcag tggcacgatc acttttcact cgagtctcga cctcttgggc 66900 tcaagcactc ctcccacttc agtctcccaa gtagctagga gtacaggcgc atggcaccat 66960 agccagccag tttttatttt tttattttgt agagttgagt tttccctatg ttgcttgggg 67020 tggtctcaaa ctcctgggct caagtgatcc tccagccttg gccttccaaa gttctgggat 67080 tataggcagg aaccaccaca ccaggcacta cactaaacta ccttagactt ttctagatag 67140 agatataaca gagctattct gaatctgggt gactgccaag atacagccta aatattagaa 67200 gatttccccc ccaccccaga atttgagctt taaagatctg ccttctggcc aacggaccct 67260 tttcttgtaa gtggtcatag aataaataaa tatttgaatg aatgagtgac tagaggggta 67320 tacttgtgag tgagctaatt catatcatgg accatatgta tttaatggaa ataagatacc 67380 tttatatttt atgtgatgta catttaaata gtctttatga taaacatttt acttgttttt 67440 taatttatag tattttatag attggtcttg ttaacataca tttcatattt ataactgttg 67500 tagcttaaca agattaggct cactattctg aggtctgatt atgggaaaga attagagagt 67560 ttaaatctaa ttatatttct ttgcattttt aggcccttga tgagcctccc tatttgacag 67620 tgggcactga tgtgagtgct aaatacagag gagccttttg tgaagccaag atcaagacag 67680 caaaaagact tgtcaaagtc aaggtacagt atttatagat ttcataaatt gtatgttcag 67740 catttgatat gtaaactttt atttggtgag tgtatttaag atttttctgc aaaataagtt 67800 tctaggagtg aaggttgtaa aaatacctca aaacaaagca ttttttgtta ttttaaaagt 67860 attataattg gaaagaaatt agattatcac agttggtatt ttgaatgcca tttcactata 67920 gacgtcttat tgtttgtata gtgttttaga gctggaaagg aagttagaag tcatctagtt 67980 taattgcttc attttattac aagaaaaatg aggctcggag agggttagtg atttctccaa 68040 ggccattatg ccaaattcat ggcagaattg gaactaccat agttcatcaa ttttaaggca 68100 tcaccccctc tatttcagcc ataaaattag aatgtgactt gtaattgatg gtgccttaga 68160 taacagtgaa ataaggtaca tctagttgtt gactccttat gctctgcctt tcttgaaaca 68220 tctttctaaa gtacttgagt tttgtcttaa tatagaaaaa ggagtgtaat tgcttccatt 68280 ttaataaatt atcagcactg aaaaatacca gacaagtgaa cttgaatata gacatttatt 68340 catttgtcaa taatttattg agtgacaaac cttttgcagg gaacttttag gtgggaagga 68400 ggatggggat attaggatga gattgacctg atctgtcctc aaggaattta taatctagat 68460 tagcagatac cttttaagcc attggttttt atttcaccaa ttacatagac tgtagaggtg 68520 ttgtgcttct gtggagggta tcagtctctt ctggagcttt tactgttcct atattcctta 68580 tgcatggatg tgttgaaaaa gctacctggg tgattctaat ttacactcct ggttaggaac 68640 cattgtgata agtcctttaa attaactaca aactggtagg gggctagtct tggccccacc 68700 ctaccttcag tttccacatg cttggtggtt ggtgctttta tagtgtgcat ttcctgttct 68760 ggcttcacac actggtattc cccaagcact gttttttgcc agtactatcc tgtagaggaa 68820 gagaaagcct tttatcaatg tttgtacttt ttcaggacta ctggtagcac atgtatcacc 68880 tttgtgtgaa gtagacaaaa gccctctctc ctggtggaaa gccttcttag cagcgcttgg 68940 catgcagata gcaagtttgt gccagttgga aaagacattc tctttggact ggagctgtgt 69000 cactccagga cccatgcttt ggcccacaaa gcaaaggctg gatttaagcc ccatcttgca 69060 tccttagctg cacatttcct gtgtaatgaa tgaactagga aaatgacatg tagcagtcct 69120 gccatttctt gctgtaaatg aaaggaacaa aactcttact ggactctact gcctctattg 69180 atagcacaga gtaccttgtg ctttataagt aatgaatatc taaagcttat ttttaatttt 69240 ttcggctgcc ctaaaaattt gtttctgtgg aggtgatgtc cattaaaaca catacacaag 69300 gaaaaagaaa gtgtatttca ttcagtgatt ttgtcagtag atatagggat atatttttta 69360 acatgtgtaa tttctttatg attcaaagtt aatatatttt taacaaattt gttgatatgt 69420 tttcactacc tgttttcatt tgttgaacat ttactatgtg ctaagctcta tctaaaggtg 69480 aaactctagt gttaagacct agtctccatc tgtaagtagc ttatagttta agtgaagggc 69540 acagatatct aaacatgtga tttaaataaa aagtgtgata atgcttttgt atgacacaga 69600 aatgcctggg atgctgtggg tataaggcat ctcctttcat agctggtttt tgagtgggcc 69660 tgcctgttac ctaaaataat tatcacatat acctgataaa gaccatgtta atgattcaat 69720 ccctataaga actcttttcc cttgttagag aaaacaattg ttttctatac tgagtagtgg 69780 aagctatctt gtttttcacc agctaattaa gtatttaaat ttgaactcat tattttaact 69840 tactcttttc acctctggtt tcccattctc tgatttctgt aatgatatag agcacagtaa 69900 agttctggaa ctagactgcc tacatttaaa tatcagctcc accatgtcta gctctgtaaa 69960 cttggaccag gtacttacac tttctgtttt ttgttttgtt ttgttttgct tttgagacgg 70020 agcttgccct gtcccccagg ctggagtgca gtgacgcaat ctcggctcac tgcaacctcc 70080 acctccctgg ttcaagtgat tctcctgcct aagcctccgg agtagctggg attaacaggc 70140 atgcgccacc atgcctgggt aatttttgta tttttaatag agatggggct tcaccatgtt 70200 aaccaggctg gtcccatctc ctgatctcag gtgatccacc tgcttcggcc ttccaaagtg 70260 ctgggattac agggatgagc caccgcacct ggcctgtttc tcagttttct tatcaacaaa 70320 atgggaacag tcatacctac ctaatagggt tgttgtgaag attaaatgtt tggtactaga 70380 gactgatgct cagatctcag ttaagtgttt gctgctactg tttattatta aggtaggcct 70440 tcacaaaatg atataatacc taatagccta aaatccccat atctttcttt ctcatttttt 70500 aggaaaagca attttacaac taaattcata actgatagta ttgaaataaa ggatgatatg 70560 tgggaccagg cgtggtgact cacacctgta atcccagcac tctggtaggt caaggcgggc 70620 ggatcacttg aggttgggag tttgagacca gcctggccaa tgtgttgaaa ccccatctct 70680 attagaaata caaaaattag ttggcctggt ggtaggtgcc tgtaatccta actacttgaa 70740 agactgaggc aggagaatca cttgaaccct gaggtggagg ttgcagtgag ccaagattgc 70800 gctactgcac tccaacctgg gcgacagagt gagactgtgt atcaaaaaag aaaaaagtta 70860 aaaaaaaaaa aaatgacatg tggaacatct agtaatttag atgtgctatc attgattact 70920 tttatttttg aaaacagata cggtctgagc agttgtctgt aaataatttt ttagttaatc 70980 tacttaggga ttgggatgtt gtataaacta ggcagctatt attttatatt tcttttacta 71040 caaatgaata attggtgcat tgatgagctt gtgttgcttg gtttattttt ggtggctaat 71100 taaacttata atctctaggt gacatttaga catgattctt caacagtgga agttcaggat 71160 gaccacataa agggcccact aaaggtaatt catgtattca ttgttaattc taatggttgt 71220 ttgggaaaaa ataatcatac ttggtattaa ttcattggct cttttgttat tgagttgata 71280 aattcaatat gtaattttct ctaaaaagac taatagaaaa aatagactta atctcagtga 71340 ctaaggaggc tgaggcagga ggatagcttg aggccaggag ttcaaggttg caataagtta 71400 tgattgtgta cactgcactt ggggacagca tgagatgctg tcttttaaaa aaaagattta 71460 atattaaata atacttgata tggtagtaaa accagtttat tcagaaataa aatgagtatc 71520 aaagaagcat tgactacatt tatttactta atccaccata tttccattac taaaatgatg 71580 gattttccaa aattaataga tgatgaacta gggattagtg atgtcagttc cacgatgatc 71640 tgaaaagtga gaaagttaga aaaccaaaaa aatcggagct cttacttctt tggagccact 71700 actggcaaat gaaatctttt aagggtcatc agcctttaca ctaacagttg gagagtttta 71760 tttctttgca ttcatttttc ctcattggca agtgaaataa atgctgcttt gctttttgcc 71820 agtgagcaag ctaaaagaga atgtgtgggt aaaccactat gtgctctttg gctggtttag 71880 ggccaaaagg aaaccccaga gcaattttta aattgtccct atccttcctg aggttccttg 71940 aaagaagagt ggaaatacgg ggcaaggaca gtcagtttca cagatggtgg tactcacggt 72000 gtagtctact gaccctgggg attcccagga ttcttttggc gggggttgta agggtaaaac 72060 tttataatag tacttaggta ttatttgcct tttttactac attaacattt tcactgatgg 72120 tacaaaaata ttgatcggta aaactgctgg cacttttttt tttttttttt tttttgaggc 72180 agagtcttgc tctgtcaccc aggctggagt gcagtggcgc gatctcggct cactgcaacc 72240 tccacctccc aggctcaagc aattctcctg gctcagcctc ccgagtagct gggattacag 72300 gcgcctgcca ccacgcctgc ccaatttttg tacttttggt agagacgggg tttcaccatg 72360 ttggccaggc tggtctcgaa ctcctgacct caagtgatcc acctgcctca gcccctcaaa 72420 gtgctgggat tgcaggcgtg agccactgtg ctcacgccaa ctgctggtac ttttaaatga 72480 agtcaatagt cattttattc ttcaccatca tgcactcagt taaaagaaaa caaacccagg 72540 ttttggtttg ttaatttacc ttttaaaagt ttttctaaca tgtttaaaaa tactttaggt 72600 aggagctatt gtggaagtga agaatcttga tggtgcatat caggaagctg ttatcaataa 72660 actaacagat gcgagttggt acactgtagg taagaaaata aattttcttt taaaaatgtg 72720 tttttagtta cacaaagaaa acttaataca aataaaatga aatttcctgg attcacttct 72780 taatacccta gatrttatta ctgttactgg attttatctt tccacatctt ttaaaaattt 72840 atttccaaag tatatgtatg tatatgtaac aaaaattgaa attatttaat gtgtatatta 72900 tgcaattgga attctagctt tttttacttc aaagtatatc ttaggtaaat ttcttttttt 72960 ccccaatctt acctattttt aagtttatag ttcagtaatg ttaaggatat tcatgttgtt 73020 ttataataga aatgtctttt aactgtgtat atatatatat atatagagag agagagagag 73080 atatatctat ctatctatct atctatctat ctatctatct atctatctat atagctccca 73140 gcaggaaaca aagtagtagt gtattttata atagttgatg tgctagttta ttttttctta 73200 ctgatgaggt ttccagtttt cttactatta caataaatgc tggattgaat gtctttttta 73260 cctaattagg tacttaatta ggatacctac tgtatcctag tatttcttta gtgtacatac 73320 tgagaaatag aggttgaaga aaaatatgct ccaaaaactt tagtagttaa tgaaaactgc 73380 cttcctaaaa gacactgtct gttttctcaa tccttgtcag attttatgtc atttaaaaaa 73440 attttttgct aatttggaca taaagtagta tctcagtttt gtttcagtat gcccttccca 73500 tgattactag tgagattgaa catcttttca tatgtttatt gaaaggaaca taatttcatt 73560 tgtgttttct ccgttttgag ttgctggctc ttttattctt tccctttttc tcttgagatg 73620 atttatcttt ttcttattag ttttggtata tgtgttcttt atatattttg gatattaact 73680 gtttactact tcagttataa atatatttct cctgttgctt tattcttgtc tctgctttta 73740 attttcatgt agtcagtttt tgtatggtca ccttttattt tgtaatttta gttttgaatc 73800 ttaaagcctt ccatcagtgt tataaatcta ttttctaatg tattcttcaa attttttata 73860 ctttttaaat ttatttttat ttttgtttta caaacagatt tttatttcac ctacagtagt 73920 atgtattttt tccaacattc taaaataatg attaaaaagt atttgtccta gattggtagc 73980 catttattcc agtattattt attaaaggta acaattctta ccatttaatg acctcagaga 74040 tgaaagtaaa cacctagtaa aatgtctttc gtgatttcag agtttgatag aactttcaga 74100 tagaataggt gagttttgtt tatacttctt tggtgtgaga ataaaacctg gtgaaaatag 74160 agggaaaatt atttgatttt gagacataag gaaatatttg actttatgta agtgagaaga 74220 ctgtgagcaa ttctatgtag gaagtctaga tggtgattgg ttgtaatagt ctactaaatg 74280 tgacacttat gttgtatatt ttagttacat catttaaaga tgtactgaaa attatttatc 74340 cagtttcaca taatgtaact tgttatgtaa cgtataagaa tctaatttta gttcagccag 74400 taaggaaagg gtattggctt tcttttaacc attaatcatt tctcaataaa cgtgagatcc 74460 tgttgagcat cagaaaaaga aaaggaaaga agagtatcta attttagtag gtaggcagaa 74520 aatgtaattt ctaaaataga gatctggtaa cattatttaa aacagagtta ctgtcttccc 74580 atgatttcta aggaatgtag catatcccta aattatttat gtatatcaca gatttatgca 74640 tataatacaa atagtcatac attccatatc tgttaactta tggtggtttt gctggggtgc 74700 tttaagtact ttatttttat taaaattttt cccctacttt attgagatat aattgacaaa 74760 atcatataaa tgtatatgta acatgatgat ttgataggca aatatattgt gaagtaatta 74820 ctgcagccaa gttgcttaac acttccatca ccttttaata gacattttag tattttaagt 74880 cgctagtttt caactgggat gaatttaccc ccagggaaca tttggcaatg cctggagctg 74940 gagacatttt aggttgtcac agctgggaag ggggtgctac tgacatctag tgggtagagg 75000 ccaggagtgc tgctaaatat cctgcagtgc acaggacaag cctgccacaa cagagaattt 75060 tctggtgcag tatgtcacta gtgacagtgc taagggtgag aaaccctact ttaaataatg 75120 aaaatacagt tattttttta aaggggcagt ctgattgcat atacttgtaa tctttcttag 75180 gtaaatctgt caatatctgt cagtaggaaa acttggcatt tctttaccat tttgagaaga 75240 gttaattagc atttaaatgt tttcctcttg aaataccaac ttcttatttt tatttggtat 75300 atcctgtttt atacaagatg ttttcatttc atatatgttg tttctttttc actagaaatt 75360 ggaaaagtta ctttaataac tctatatttg aagttactca ctagaaaaca taaaatgtga 75420 attgaattcc aaatgtagtc ttaaatagta gatctgtttg cactcagaaa attgtaatac 75480 atgtcatctc tttgatcttt gaaaaaatcc ttcagtagct tccattttta ccccctctca 75540 tagtttttga tgacggagat gagaagacac tgagacgatc ttcactgtgc ctgaaaggag 75600 agaggcattt tgctgaaagt gaagtaagtc atcatttaac aaatgaacat gtcttaatat 75660 tttttattgg gaggaataaa tttttgcttt ctcaactcag taaacctact tcctaatcag 75720 gaaagttcat taatacacaa tacccttatg atgtaatctg tgaagcctgt ttaaggccct 75780 gtgtttgagc accaggttct gtcttctttt ttgtctatgt aaagtgtttc agtatttgtc 75840 cacagagaaa atacaatggg ctttaattat tatctttagg cagtttttaa acagacttaa 75900 aatttgtaaa taactagaaa acagatttct gatttgtaat ctacttgatc cttgtcgtag 75960 ttaaaggatg tgtagataca acagtcttta actactgcta ctgctattag ctgatattat 76020 tatgcccttt cctcttgcct gactgccctt cctaaaattg tcccttccat tccttatcat 76080 cctttcagat tgatttaggt cttattccct aacatcaaaa tatgagtaca taattattta 76140 ttgtctgcct tcctcactag aatgtgtcac acggtcagag actttgtata tcttcttcat 76200 ggctttgttc ctagttgctg taacagtgcc tgatgcagag tatgaacttc ataagtgtta 76260 gttggatgaa tagatgaggt gcttataatt gccagggatt ttgctaagtg ctttacatac 76320 attattgtat ttgcaacatt atgaggtagt tactattatt atccccaact ttcagattag 76380 gaaatggata gctagagctg gttttctaac cagcactttc tagcaccaaa gccatatcct 76440 taaattgaaa gatgttacta agagcagagg gttcacaaag ttttgtaact aactcgcttg 76500 caattcagaa tttaaaaggt agatgttgct ctaaatggtg ttacgttctc taatccagtc 76560 tacagatatt cattgataca cagtgtggcc gagctatttc ttttctctac tcactatttg 76620 gaatagtgtt tctttgggga tattttaata ttactaatta atattactta tttgttatta 76680 tttattactt taatattact ttaatattac ttatttagta gtgttattac ttaaacacta 76740 tttggaatag tgtttctttg gggatatttt aatattactt attaacctat tagactttat 76800 gtttttgttt tgaaaaattt ttgcttctct taatgattga attcccactt aaatttagaa 76860 tttggacagg ttctctgtat tataattcca gcctatccct ttagtctcat attcaaccac 76920 ttccctgtac ttctgcctta ttagactgct tggtattttc agactatggt ccatactttc 76980 ctttattgac tcctttcctc ttgttggaat ctcacttatg ttgaaggcca ttataaatgc 77040 ttcctcctct atgtctttcg tgagctctct acccaaattg gtcgttttgc acttctcttg 77100 cttattcttg ttttaggacc tttttttttt tttaactttt ctcatttttg ctgtaagatt 77160 ttaagctgtt ttaggggaat atcctttctt tcctcacttg ataggccccc cttttcctag 77220 caatttctac acaaaagcct tcaataaata tgtgtggaat aaactgaagg tttacataca 77280 ataaatacag acagctttgg ggcaagtata gtgataaagt acagaattct ccctgagttc 77340 gtggaattat tttaatttca tttccgtaca gtcagtgagt atgaggactt gaagacatgg 77400 tttttctcct gttaagtgaa ggtctgcatt ttattttttt agtctagaga aatagccaaa 77460 atattatttt ttttaaccag gagaattttc agttggaatg gttcataaga cagttgtaag 77520 ttaaatgctg aaggtaatat gaacaagtgg ctatgtatcc ttcatatctt caaggaggtt 77580 cagttttagt gtgtctgtgc aggtgtgtac ttctaattta tgttttgcta gaaggaaatt 77640 agaatactgg tgtgtgattc ttcaattccc ttcccatgtg cccggagctg tgtagtgcta 77700 ggtaccggga caaaggttaa gccaacccat gtaaacatgg aacttatctt tgggtactta 77760 ctccctgttg gaggagaaaa agcattaatc aaaccaagcc atgtaaacat ggaacttatc 77820 cttggatact tactctccgt tggaggagaa aaagcattaa tcaaacaatc aaacatattt 77880 taaaatttga gtatattaag ccctgtgaag atgtggtcca gagtgcaccc aataggggaa 77940 tttaatctac catctgtatg ttcttttctc tttatttggt atttttcata ttgttttaaa 78000 agttttaacg tttttgaaag accattcaga gtattagtag tagtagtagt ccataacatt 78060 gtcctttaag aggaaagctg cttcatactg ccagtatttt gggtcactaa tcccatttaa 78120 taaactactg aaattaatcc catctcataa acactgaaat tcaggatttt catggaaaga 78180 gatcacattt gcacttcagt cagtttatct ttttatttta ttttattatt attattatta 78240 ttttttgaga cggagtctcg ctctgttgcc caggctggag tgcagtggtg cgatctcggc 78300 tcactgcaag ctccgcctcc caggttcacg ccattctgct gcctcagcct cccgagtagc 78360 tggaactaca ggcacctgcc cccacgcctg gctaattttt tttttttttt ttttgagatg 78420 gagtctctct ctgtcgccag gctggactgc agtggcacga tctcggctca ctgcaacctc 78480 tgcctcccgt gttcaagtga ttctcctgcc tcagcctcct gagtagctgg gactacagtc 78540 gtgtgccacc acgcccagct aatttttgta tttttagtag agacggggtt tcaccatgtt 78600 ggccaggctg acctcgatct cttgacctcg tgatacgccc gcctcagcct cccaaagtgc 78660 tgggattaca ggtgtgagcc accgtgcccg gccacgccca gctaattttt aaatatttgt 78720 tagtagagac agggttttac tctgttagcc agggtggtct cgatctcctg acctcatgat 78780 ctgcctgcct tggcctccca aagtgctgag attacaggct tgagccacca cgcccagcct 78840 cagtcagttt aaattccaac actgaagcca ctggttttag ccataaccag cttaatgagt 78900 atgttacctg ctgcctaaca tctgtaggac agtttaggta tttctaaaga ccattcatac 78960 tttgagtatt atacaaaagt agattcatca tctgatattt atttatttat ttattttttt 79020 gagacagagt cttgctctgt cgcccagact ggagtacagc ggcatgatct tggcttactg 79080 taacctctgc ctcccgggtt caagcgattc tcgtgcctca gcctcttgag tatctgggac 79140 tacaggcgtg cagcaccacg cctgactaat ttttgtattt ttagtagaga tggggtttcg 79200 ccatgttggc caggctggtc ttgaactcct agcctcaagt gatccgccca ccttggcctc 79260 ccaaagtgct gggattacag gcgtgagcca ccgcgcccag ccaattatct ggtattcttt 79320 ctttgtattt actatgtgca attgaaaact ctagagaagt ttaggtaaaa tcccagttcc 79380 atattttctg cagcccattt taggtaacag ggccatagac attttccctc taggcctacg 79440 gagttttcta tgtgccttta atacagcttt caggagcctc tggtttttgc tctttaggtc 79500 taagatggga caaattctac cctaccccca ggtcttttta gtcccttgaa acaaacatgc 79560 ccctactttc ctggacattg agatagggac agagatattg cttatgtttc ttcttgttat 79620 gtactaggga gagactgtgg tctcttattt gaaaattact atgtgtgatt cttaaacaac 79680 tctttagaaa ttttctttcc tgtagcaagt gaaatgacta acagatactt aaaatagaaa 79740 tcacactgtg tgatataaat ctctctctct cttttatttt tctgctttaa agagagcaac 79800 cttaattttc ggtggggaga gcatacattt aatccatctg tttgcttgac agaaagtgaa 79860 tccggattaa tttattttcc ctgtttacca aataaagtca tttagtaata ctctatagtg 79920 atacagtatt accaaaataa tatttcagat tacatagaaa ttttcaccat aggagttaat 79980 gacaggcaac gtctagattt tccgggtggg tgtggtggct catgcctgta atcccagcag 80040 tttgggaggc cgagttgggc agatggcttg agcccaggag ttcgagacca gcctggacaa 80100 catggcaaaa ccccatctct acaaaaaaca aaaattagct gggtgtgata gtgcacacct 80160 gtggtcctag ctacttggga ggctgaggtt ggaggatcac ttgagcccag gaggttgagg 80220 ctgcagtgag caatgatcat gccgccactg cactccagcc tgaatgatag ggtgacattc 80280 tgtctcaata aaaatagttt ctgtttggta tatactgagt ttaggacagt ttttgcctcc 80340 aaataatttt tatttttatt ttttgagatg gaatttcgct cttgttgcac aggctggagt 80400 gcaatggtgt aatcttggct caccgcaacc tctgcctgcc gggttcaagc gattctcctg 80460 cctcagcctc ccgagtagct gggattacag gcacccacca ccatgccagg ctaattttgt 80520 atttttagta gagacggggt ttctccatgt tggtcaggct ggtctccaac tcccgatctc 80580 aggtattcca cctgcctcgg cctcccaaag tgctgggatt ataggcgtga gtcactgcac 80640 ccggcccaaa taatattttt taaatgcctg atttaaggtt cattaggaca gggttaggtt 80700 ctgtacaaac taaatttgaa agattagatt ttttgcccta tgagagttgt gaaagctatg 80760 atttttgtca gtcttttgaa ggaatcaatc tttagttaat cctttatggc tagtaccttt 80820 tttcttgatc atactgttcg ttgttagata catacatcta tgtaccttaa acacatttac 80880 atatattcta atgaattatg taaaaaagtg ttaatatggt tcatcttttc acagttttgt 80940 tcattgtagt tataacaaaa ttgtctcaat tttaaaaaag ttatataaca tttcttttta 81000 catattacat tagtttagat gcctgctctt taattctaag ttcatggata ctctttctta 81060 taccaccctg gtttgtatct ttaagaagtc tctgtcttaa atagttcctc acttttattt 81120 tcatctgggc agttttcttg gaaatctcta gttgctctga tgtttgtgaa attatttata 81180 acaggaaaga ttcttcttag gatgaggaaa ggaacagaaa ctgtggaaaa tcttaagaaa 81240 acgtaggaaa ggaaaaaaat caaatgtaga aattaaatgt aatttaaaaa attaaattaa 81300 tgttacattt tattaaaaaa ggtttattgc agaaaatgtt tgcagagtag taatataact 81360 tactccgagg ctttttgttc gtgcgattga tgctttatct tcataattaa ttttgtttct 81420 tattaataat tttattttat ggattatgat tccccatgta caaattaata ttggagctgc 81480 agttggccct aaccttacaa atagatttct taattctcac atttgtcttt ccactgagct 81540 gtgtttgttt tgtattttaa tcattcttag atgcctatca accttcacat aggtaatgaa 81600 tcatctagag agagcagcca aatttctttt cacttttttt ttccagtaat gagctcattc 81660 atctttattc taatatcttt gaaactagaa ttttctatct ttaggaaagc tgtgttttag 81720 cttccctggg gggagagttt aatctcaaaa tctgagcata ttcttaaatt gatttggttt 81780 aaaacaaaga aggcacttgc aaagtgttaa ttttttgatg ggtttatgtt cttacccaca 81840 gacattagac cagctcccac tcaccaaccc tgagcatttt ggcactccag tcataggaaa 81900 gaaaacaaat agaggaagaa gatctaatca tatgtaagtc cattttcatg actgttagtt 81960 gaacctgaat tcatttctcc ttctagtatt gtttatagtt attttaaaat tgtaatttgc 82020 aaaatgctaa ccagatctaa tagtaatttc ggatctaata gtaatttcag actcctcagc 82080 atgggtgatt gaggttctcc atgctcagcc aaccttactt aacagctgta tctgccttca 82140 atttctccct gcatgttatc ttgatgctgt agtcaaaatg aaaccagtca ataattcctg 82200 aatacatcgc ttgttttcta atatcatgct tttattcatg ctattatttt tggtctccat 82260 gcctcccttc ctggtaactg tcttgtccat gccttaattc tcaacaaaaa tgcttaaacc 82320 tttatgtgaa ccattttgtg tctccccaca ctttccttga aaccagagat gtttctctcc 82380 cctgtaaacc caattctctg gcatagtatg aactgtggag ccagttggtc tgggtttgaa 82440 tcctggctct gccacttcct tgctgtgtga tcttgggatg gttacttaat cttttcatac 82500 ctcacttcct atctataaaa tttggataat aatattacct atctcttagt gttgttgtga 82560 aaattaagtg aattcatatc ctttaagtgt ttatagagta aatgttacat aagtactaga 82620 taatcaacat catatactga catgaaaaat ttcatgatga aataccagta tgtaacagtt 82680 tcatttatat atgaaaatgt gtttacatgt atatataaaa attctgcttc ctggtgttta 82740 tgtctgacaa ttaggactgg ttcttgctga attattcgaa ctttaaaaaa tttctacgtt 82800 tttatatata ttataggaaa aatagtctct tatttgtcaa taagtttttt taatttttag 82860 aagattgcat ctagtattta gacatatttt tacgatttta tacacatcat cctaaagtgt 82920 aaaaataaat ttaaaaatga atttattgtg caatatcacg taatattgac ctgcaaatgt 82980 gactaaaaat tatgatctgt taaaaagtca gttatttttt atttcctctt ctgtcgttcc 83040 ccaactgtta gatgtttctt agttgaggcg tccgttttgt gaatatgaga ataaagcacg 83100 catttgttgc tgaataagtt gatctttgct ttctaaagac tccagcaaac attctaagtt 83160 agtggttttc ctgttgagaa tctctatgtg taatgagaga tttgctgttg agattgtgtt 83220 atacatgtgt aaagtatgaa tgcagacaaa aggttgaaga ttactagtgt aggaatgaag 83280 acctgctcca tctgtaatct tgttttgctt gctcttcctt gcccttctct accacggttg 83340 gtcattctca ctgccaagga acctgacaat gttactcttg cttggcgttt taagccttgg 83400 tgtgtaagca ttacaggcaa gtactacagt ttgaaatgcc catctccttg attacatttc 83460 ctcaaagttc tttctcccct ttcttcaaag tgattttctg caacaaagtc ttaaacctag 83520 atcttaaggg cattcatctg accttcattt cctcttccat tactccattg aaaaaagtct 83580 ttgctgtgat ctcagattgc catttaaaat gtatttttgt acaaagatat gtaaagttat 83640 ctcttcttag gattcccagg acttcgttgt tggagtgatg tatcaccaaa tttgttttct 83700 tttagcagct gttattccac gtatcagtta atctacccct agttattcca aaattgatta 83760 cttttaacat cttccattaa attgagtttc agtatattct acctttaatc ctagtaagtt 83820 caagtatttt ctttttttaa tgattatcat cctctagcta aatttttttt aaagcaagtt 83880 attgattctt ttaatcaatg tattactgga ttgaattttc tccttataac ttacccattg 83940 tttgcagcga ttgtgtttaa gtcatccaga agaatagaat acctgaaatt atggaaatac 84000 ttgtttgtcc ctctcctctt gaagttgttt tcccataatc tggcaaaagt ttatgtgcaa 84060 ctgctacaat aggaaagcag tctatttgct gttatatttt tccccttcag aaaaaagata 84120 aagcataagg tttacaatga atcttgttaa caagggatta tttatgagta tggattttac 84180 ccagtaacag tgctaagatt tacccaatac ctggtttata cttgctattt aaatacaggc 84240 ctgcagatct gaacacattt ttaaattagt gtataattca tgtatcataa aatttactca 84300 tatattttaa tctgtagatt tgtatccaat acttcaccct tttaaagtgt acagtttagt 84360 ggtttttagt atattcataa aattgtgtaa ctatcatcac taattccaga agatcttttt 84420 attttgccgc tttattttaa tatgagaatt tgattcattt cctccttgat aaaaattgag 84480 aaaaaaatta aaaccctttt atagttaagc gtacataggt tctgtgtgtt tctaagaagg 84540 tatgtatacg ttgtcagtct tccagatact aatagaacat ttcaatttac aaagtttgaa 84600 actgagtaaa ctgatttgcc taaaataaaa aataatcaga attgcatgtc aaaggttgag 84660 cctcttgttt ctagctcaat ttgtatcatt atttggtatt ccttggccca tatcttgtct 84720 aaagttcgtc tttcttgatt tacaactaat tagagttaat agtgaagtgt gatcagcttt 84780 aagtgattgg ctgttagagt attatggtaa aaatttccat gtaattagct ttctttctag 84840 tgacatcatc agattttatt tcatagagac agcgcattat tttgttgtgg ctctacaaat 84900 accttgccac caaagaaata acaatgtcag aaatgaataa aaatcaagca ggctctctgc 84960 ttgctctgtc tttgctcctg taaaatattt tggatgctca ttaattttag ttcattagtt 85020 tattgagtac ctgtgtaaac agttttctgg ctaagctcct ctaaaaggaa aagttaagga 85080 gcttcatgtg taatagtatg gtttacactg atgtatacat tttgtgattt tagaataaaa 85140 atcagaaata ccataataaa aaaataaatg catctttttt attttatgaa caagtattta 85200 tttagcatct atactgtgcc aagcattgtt caggtacttg agatatatca ataaacaaaa 85260 aaaaattact taaaaaatct tcaacttgag agcttacatt ctctcagggg aggagatagc 85320 gaattaaaca ttggattcac atacttggtg tgaaaaaaaa gaataaacat aagaaataag 85380 taaattatat agtatgataa aagtgaatgc tgtggaaatg aagagcaaaa tgaaaagagg 85440 tagatgggca gggactgtaa atttgaataa ggtggaacaa agtcttgagt aggtgagggg 85500 gaaggggcat ctagtctaaa aatggagatg attttagaca ggaaccttgc aattttatat 85560 gtaaggtgga tagatgtttc agctagctga acaaaggtgt tttgtttttc tttttgatat 85620 actgtgaaat ctgaagtctt aacacttgtt aagtcttaag aaaagttaca atttttacaa 85680 tgatgcagat aattttgtta gtcaaataaa gcccaatttt ctctatgttg tacagatttt 85740 gatatgtgtt atctctgaca ttgtagcttt ctagctgcta ctgataatag agtgttcata 85800 aacccctagc tgaatgagtc ttctgtggct actttgagaa agcagtaatt atgattttaa 85860 gcttggttgt atcctgtagt cattagacag taaaaaaagt cactactctt aagctcttgt 85920 aaatcaagta gaaatcagac ttgtgaggac tgtcgttact gtcccaagct gtgtgtctat 85980 gtgtagggtc ttattctcca ctaatgtaaa ggtggttgag ttttcttatt gttacctact 86040 ttctaagtag agattgaact tctttaagtg ccattgaagt acaatacatc tttttttttt 86100 tttttgagac ggagtctcac tctgtcgccc aggctggagt gcagtggtgc gatatcggct 86160 cactgcaacc tccgcctccc gggttcaggc aattctcctg cctcagcctc cagagtagct 86220 gggattacag gcacccgcca ccatgcctgg ctaatttttg tatttttagt agagacgagg 86280 tttcaccatt ttggccaggc tggtctcgaa ctcctgacct caggtgatcc acctgccttc 86340 gcctcccaaa gtgctgggag tacaatactt ccagccaaag tacagtactt ctattcaata 86400 gcaagctttg aagatttgct ttgtttctgc cagggaacaa tttttaagtt atttgactga 86460 tcattgaaat caagggggtg gtattcccca tgtcacttta aaaagcttaa tattattata 86520 atagttttta cttttttggc caggtggggt ggctcacgcc tataatccca gcactttggg 86580 aggcgaggtg ggcggatcac gaggtcagga gatcgagact atcctggcta acatggtgaa 86640 accccttctc taccaaaaat acaaaaatta gctgggcgtg gtggcaggca cctgtaatcc 86700 cagctactca ggaggctgag gcaggagaat tgcttgaacc caggaggcgg agcttgcagt 86760 gagccaaggt ggtgccactg cactccagcc cggcaacaga gctagactcc gcctcaaaaa 86820 agaaaaaaat agttttcact ttgtttacat ttatattgga atatataaat ataaaggcat 86880 gacggttact ttgtcatact tttggaaagg catcaaagac aacttcagag tatactagat 86940 agtatactaa ataggtaaat catgtttaaa gttttttagt agtttaatct gtcagttttc 87000 cttttttggg cagtgaaact gatcattgca aactatctga aaaacacttt tatctttgta 87060 ggtattcaat aatagccatt ctaaaaagga catatgttgt tgtccatcaa acatttccta 87120 gtctttactc attctgggca ttttctttgg tgctgattct ttattagaat cagattcaaa 87180 ttggaatagc tttttgtttt gttttccttt ttactacact ttcctcaaac atcaagttga 87240 tggtttcaga tacttccttt ttcttctgac agttatcttt ctaatttact agtccaatat 87300 ttaatgttac tgaaaggatt ttctgttttt cttttaattt tgaaagtttt tagcatcata 87360 caaaagaggc ttattggaca ttttatgaat acctaattgg tgtgtatttg ttgttatgga 87420 tagcagtggt gatctctttt cagattgcct cctcttccca acctaacctc agttaataaa 87480 ggtctgctct cctacctttt aaaattaact ttgtactgga gatatagaat tactgtctcc 87540 agaattcctt taatttgaga ttgggattgt gtatatttta gtgtaatact gggatgttta 87600 atatggggcc ttcacctttt tgaggatttg ggaaccagtt atttgtcctg atcttcctcc 87660 tcaatttttt acagtagcac taaataaacc agaaatattt ttgggaaaat gatgtgcttt 87720 cctctcatga taaactcttg ctgttatttt ttagaaattt agaaaatgat ttaagattta 87780 aattagccat tttacaggtc ttaggaaatg taataagagt ttgggaatac ctatattcat 87840 cattggcatg cttattcttt gtgaggataa atggaaatat tttatttttt tctctcccta 87900 gaccagagga agagtcttca tcatcctcca gtgatgaaga tgaggatgat aggaaacaga 87960 ttgatgagct actaggcaaa gttgtatgtg tagattacat tagtttggat aaaaagaaag 88020 cactgtggtt tcctgcattg gtgagtagct tcagtataca agagtttaat ttmaaacttt 88080 ataagtttat gaagaaaaca attttcttac taatgttttt cataggtaca aaatgaggaa 88140 atgtttttag cattatttag ttcacactaa gcttactttc aacgttgtct ttgaatacaa 88200 agaactcttg atgtcattca ggaaaggcaa aatcttgata atcttagagg tttagctatg 88260 tggaagttga attctctctc tctcggtaga aaattcatga tgcaaacttt taaagctcct 88320 aagtccatgt aggtctagat tgcaaaagaa caacagaatc tttcttggtt gacagggata 88380 actagacctt ttcctcatac ttagtgaatc agggtgtttc catccttgat cttaatcttg 88440 ccttttaggt actccctata taatcatgac tgcaattata cattacccca ctcttcaatt 88500 ttctctacag tagattgtta attctaccta ctttttaatg tttttctctc caaaagattg 88560 caattcctta gattccaggt tgaatatttt gcattttttc cttgttttct cacgttgcct 88620 ttttgtgatt ctgaggaggt actcaagatt tattatcagg agtatcctca tgattgtccc 88680 aagttcttgt atttcaaact gttaagacat gacagaaagc cttttgagct ttttatctct 88740 ctgtttttcg taaccactag cttctttagc aagttgaaag ggccatgtat agaccatttt 88800 ctttgacttt tagaaaggga acatatacta atatctttca cggacataac taggctgctt 88860 ttcaaataat cttttggtag acaaaataat ttgtagctct taaagcaaat agaggttcaa 88920 ctaataatta gctatatatg atactttaat gaagatttaa ctctgtggca ataagaatgc 88980 atttttttct ggacagcata taattggata aatcacttaa ggttttaaat ttcaagtagg 89040 agactgaagt taataatcag tatctcttaa gaattatctt ggctggggtg tggtggctca 89100 cacctgtaat cctagcactt tgggaggccg aggtgggcaa atcccctgag gtcaggagtt 89160 cgagaccagc ctggccaaca gggcaaaacc ccgtctctac taaaaataca aaaaaattag 89220 ctggctgtgg tggcacatgc ctgtaatccc agctaggcag gagaatagct tgaacccagg 89280 aggcggaggt tgcagtgagc caagatcgcg ccactgcact ccagtgtggg cgacagagca 89340 agattccctc tcaaaaaaaa aaaaaagaag aaaaaaaaag aattatcttg aactctttaa 89400 aattttgata tgtctgaatt gtcctatgat gattttatct gaagaccatt gagtcatttg 89460 catcaaacag tgagatccac tttttttttt tttccccgga gacagaggct tactccgtta 89520 cccaggttag agtgcagtgg tgtgatcctg actcgctgca gccttgacct cctgggctca 89580 acaattctcc cacctcagcc tcctgagtag ctcacactac aggtggagac caccacatct 89640 ggctaatttt taaatacttt gtaaatacga ggtctcccta tgctgcccag gctagtctta 89700 aactcctgga ttcaagtgac cttcccacct tggcttccca gagtgctggg attacaggtg 89760 ggggctactg agcccagccc acatctttta caaatatgtc ctcccctcac ttcatattta 89820 ataagctata aactgaagat ccgctgataa acaaaagaca ctttgaaagt actattctca 89880 cgtttgacaa ctaatttgtt ctcaaatatc gagagacaga atgggtcaaa attattctct 89940 gtctctgttt tgtatgttct cactccctta ctaaaaataa ttagccaaat tctcctctaa 90000 tgcttttttc ccttatgtta atgacattca ataaaatttt tgtgttctca agattaacac 90060 atatttgtta ggtatgcatt tatcatatac caagttctgt tcaaaatatt taacaaagat 90120 agactcattt agtggtgtag aggttttttg ttctcatgat taataaactt tccttttttc 90180 ttttcttttc ttttcttttt tttttttttg agatggagtt tcgcccttgt tgcccaggct 90240 ggagtgcaat ggtgcgatct cggctcactg caactccgcc tcccaggttc aagtgatcct 90300 cctgcctcag cctcccgagt agctggtatg acaagccacc atgcccggct aattttgtat 90360 ttttagtaga gacagcgttt ctccatgtcg gtcaggctgg tctccaactc ctgacctcag 90420 gtgatctgcc cgcctcagcc tcccaaaatg ttgggattac aggcatgagc cagcacaccc 90480 ggtgatgatt aataaacttt tctctacaat ttgtgcttga gaatctgcta actctttatt 90540 ctcattctct cattttcctg ctcattgact aacaattgca gagacttaga aatgggatta 90600 aaacactgtc ctttgtttat atgtatttgt atgaaaactt atgtccataa tatatataaa 90660 ctcttttgta tatttgtggt ttttgtgcta aactaggtaa ccatggtctc aaatgaaaaa 90720 gaaacaatag aactaccttt gtttttactg ttttctactt gtgatgtcca tttacccttt 90780 ttagtcaaca tgtcttattt ctttatctcc actgcttagt tcagtgttcg gcacaaatta 90840 artatgcagt aaatgtcagc tcttgtaatg atagtgaaaa atgaggacaa ttatttgttt 90900 taataaatat aaatatttta taataccttt gtaccttcct gtgtatccaa catttagtgg 90960 atacctatat gtacaaggag tgtgctaaca cctattatga gataggtatt attatttatc 91020 ccgatgtaga gataaggaaa ctgagtatta gcagggttaa gttagttgcc acactttggg 91080 ttaattatta agctgaaaaa aagcatcagt gtttgcgatt ctttctttct ttctaaagca 91140 ctaggagtct ttgtgagttt cttttgaaag tatgttttaa ggccagctgc agtggctcac 91200 acctgcagtc ccagcacttt gggaggccga ggcaggcgga tcacctgagg tcaggagttc 91260 aagaccagcc tggccaacat agtgaaaccc cttctgtact aaaaatggaa aaatttgcag 91320 gacatggtgg tgcgtgctta tagtcccagc tactcgggag gctgaggcag gagaatcgct 91380 tgaacctggg aggcggaggc tgcagtgatc tgagatcaca ccactgcatt ccagcctggg 91440 tgacagagca agacttttat ctcaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaatatata 91500 tatatatata tatatatatt ttttataaaa ccgttttgtg ggccaggcgc ggcggctcac 91560 acctgtaatc ccagcacttt gggaggccga ggtgggcaga tcacttgagg tcaggagttc 91620 aagaccagcc tggccaacat ggtgaaaccc tatctttact aaaatacaaa aattagccga 91680 gcatggtggc gggagcctat aatcccagct acttgggagg ctgaggcagg agaattgctt 91740 gaacacggga ggtggaggtt gcagtgagct gagaccgtgc cacagcactc cagcctgagt 91800 gacagagcaa gaccccatct caaacacaca cacacacaca cacacacaca cacacaactg 91860 ttttgtaagt ctttttctca ataggaacat taaatttttt aatattagtg ttttgagtta 91920 ttaatttaag cttctcacac cataatcact aaaaagtttc tttgacgaaa atgaaaaatc 91980 taaggatttt taatattgaa aggttagtaa tctgtttagt cagccatttc actgttgttt 92040 ttaaattctt agaattattg atgtatgtta tctcctccca aaattgagat ccattggacg 92100 gtgatggata gtttttggta actagagtga aaaattatgt tctaaatcga ccaatcttga 92160 tataatcgca caaatagtat ttgtgtttct aatttatgtt atttttataa atcgagtaaa 92220 ttcaaagtgt tttagtaaca tcataagaaa acagttaact gggtctgggt ggcctgtgcc 92280 tgtagtccca gctacttggg agactgaggt gggaggatcg attgaaccct gggaggttga 92340 ggctgcattg agtcgtgatc acgccactgc acagagtgag accttgtctc aaaaaaaaaa 92400 aaagaaaaag aaaaaggaaa caactacata gtcccaggaa ctataaataa ctaccatgca 92460 ctgagtattt accctgacta ggactgtgct gagcttatta atataagtag tattgtttaa 92520 tcctaattga gacttactct cagtctcaat tttttatttt ccaatttcat tttaagaggg 92580 ggaaatctta gattccatgg ctcatgcagt tctccttttc agtttttttc accaggttct 92640 cctcttctaa tcaccctttc aagctgtaat tcaggattca gaatgtatcg ctgtattttc 92700 tttacggtga tctcatctcc ttttaaagct tcaccttgca gagatagcaa agagataaag 92760 tgaccaagaa aataacatac atgggtgaat gggataaaat gcatattgtc gttctgtggc 92820 agaactggac agtgagccca cttactcata caccagtgtc atcactggcc aaagctatca 92880 catgtgcagt tttcgaaatc cttcgctcat cagggaaaag taatgagtgg atttggctca 92940 caagccccaa gttttagacc cctaatattt aggctgtggg ctctcaaatt tatatccttg 93000 agtccagacc tctacgcttt gctccagacc tgatttttct gttcactgtc ttcatttgga 93060 tgtcttttag ccacctaaaa cttagtagtc ccaaaactcc tcttattatc ccttccctca 93120 tgcacaaact tggccctttt ttgttgtttt tctttcacag tgaataactc tgtcatctgc 93180 ctatttgtac aaggcagaaa actaagcatc ctcttattgt tcatctttct acttccctaa 93240 ttatcttgga tatttctcat tcctttaatt tgccattgcc accactttac atccaagcca 93300 ccacctctca ctcaggtgat cacagtagct tagtttcttt gaacagttgc ctctgcgtgg 93360 tccattttct tcatagcagc caaagtagat ttttaaaagg aaaatctatt atgacattct 93420 ttggtttcag attctttcaa caaagatgtt agattttatc ctaagaatta atatggccta 93480 caaggctctg gaaattctgg acccttgttt ttttcttagc catgatgtat cccttcagtt 93540 aaggatctaa ctctaccttt tttctgtttc tgactagaac ttcgtccaca gcttcatcct 93600 agtcatagct gtgcttctgc tttcaatgcc ttgcctatgc caaaaattct agattatcca 93660 gcagctggaa ggatgtagga tgccaattta aaattgcttg tgtgtgctcc cacgtgtgtg 93720 tgtgtgcctg tttaccctca aactaaaatt agtttttaaa cttcaagctt acacttttta 93780 aaaggattta aatggtgggt acttacctat ctatgagttc ttttagttat aatagcttca 93840 ccctttttct taaaaggtgg tttgtcctga ttgtagtgat gagattgctg taaaaaagga 93900 caatattctt gttcgatctt tcaaagatgg aaaattgtaa gtataattta cttggtttga 93960 aaaaccagtt tataaatata tgctgttatt ttgaattagg atgttaaatg attaattagt 94020 tatttccaat taccttaggc ttttattttt catcatacag aatgtttcaa agagtcactg 94080 tttttcatat tatcttaatg tgttgtagtc ttatagtatg tttttcctta ctaattttgg 94140 taatcttttc caaactagac actcagtact gtctaatttg tcaacacttg tccacataat 94200 agctggttat ttgaggcaaa tttgccatat ctaatgtatt taatacttta ttgatttttt 94260 tgtttttttt gagacggagc tcactctgtc acccaggctg gagtgcagtg gtgcagtttc 94320 ggctcactgc aacctctgcc tcctgggttc aagctattct cctgcctcag cctcctgagt 94380 agctgggact ataggtgcat gccaccgcac ccagctaatt tttatatttt taatagagac 94440 ggggattcac tgtgttgtcc aggctggtct cgaactcctg atctcgtgat ctgcccgcct 94500 cgaaccccaa agtgctggga ttataggcgt gagccatcgc gcccggccta ttgatgtatt 94560 tattgacatt atttccttag ggtttgttct tgatggtcta attaggaatt tccaatactt 94620 ctactagtta tgtctaatgg ttatagtgat acctgttgga taatactcaa cttttgcagt 94680 agtctgagga ttaaaatggg ttacttgtat atggatggta agtgttaaga aaatgatttg 94740 ttaatttttc tttaagaatt atatgactag agaagccaga tattttatac agaattgtat 94800 tgggtataaa taccttaaaa aggtcatcat gttttaagat ttgatgtttg aattttcaag 94860 ttcgggaaag gtatttttat ttttaattat tttattttac tttatttcat ttttttggtg 94920 agggtctcac tctttcaccc aggctggagt gcagtgacat gatcatggct cactgcagcc 94980 atgacccttt gggctcaggt tatcctttca cctcaatttc tcttgagtag ctgggactac 95040 acctggctat ttttgtattt tttgtagtga taaggtttca ccatgttggc caggctggtc 95100 tcgaactcct cagctcaagc gatccaaatg ccctggcctc ccaaaatgct gagattacag 95160 gcgtgagcct ctgcacccag tctgaaaagt tagcttcaaa aacagtatgt aggctgggcg 95220 cggtggctca cgcctgtaat cccagcactt tgggaggcct aggcaggtgg gatcatgagg 95280 tcaggagtgc aagaccagcc tggctaacat ggcaaaactc cgtctctact aaaaatacaa 95340 aaattagcca ggcatggtgg tgggcgcctg taattccagc tactagggag gctgaggcag 95400 gagaatcgct tgaacctggg agatggaggg tgccgtgagc agaaataatg ccattgcact 95460 ccagcctggg tgacaacaac aagattccat ctcaaaacaa aacaaaacaa aacaaaaaaa 95520 aatgtagaac caactttaac aaaattggga taatcagagg aatttgagca cagaaattga 95580 atattgactt aatattaaat tgttgggggg taaggaaaat gggaaaacgt tagtaaaacg 95640 gcacaaactt ttattggtaa gatgaataaa atgtacagga tctgatgtac agtgtggtaa 95700 ctagtattct ggcagcctcc caagtagcta gcactacagg tgtgtgccac cacgctcggc 95760 aaatttttgt agggtttttt ttttttgcca tgttgcccag gctggtcttg aactcctgag 95820 ttcaagtgat ctgcccactt tggcccccaa agtgttggga ttacagttgt cagccactgt 95880 gtccaacctt tatttattta tttattttat tttatttact tttttttgag atggagtttc 95940 actcttgttg cccaggctgg agtgcaatgg cgcaatctca gctcactgca acctccgcct 96000 cccaggttca agcgattctc ctgcctcagc ctctcgagta gctgggacta caggcatgca 96060 ccaccacacc cagctaattt ttatattttt aatagagatg gggtttcacc atgttggtca 96120 ggctggtctt gaactcctga ccccaggtga tccactcgcc tcagcttccc aaagtgctgg 96180 gattacagtt gtgaaccact gcactcagct caacctttat tttttaagag atgggatctt 96240 gctctgttgc ctacgctaga gtgcagtggc acgatcatag ctcactgtaa ccctgccctg 96300 ggttttttcc agtcagctcc ctgcctcaga ctcccaagta gctgggacta caggcacatg 96360 ccaccactcc tggctaattc tttgcttact tttttagttt gtctgtggag actgggtctc 96420 gctatattac ctatcctggt cttgaacttt ggcatgaagc agtcctccca ccttgacctc 96480 ccaaagtgtt gggattacag gcatgaatca ccatggctgg ccccaaacat tttatcatta 96540 tattgccata aatcgttttt ggccgggtgt ggtggctcat gcctataatc ccagtacttt 96600 gggagactga ggtgggtgga tcagttgagg ttaggagttc aagaccaacc tgtccaacat 96660 ggtgaaaccc tgtctctact aaaaatacaa aaattagccg ggtggtagtg gcatgtgcct 96720 gtaatcccag ctactcggga ggttgaggca gaagaatcgc ttgagcctgg gaggtagagg 96780 ttgtggtgag ccaagattgt gccactgcac tccagtctgg atgacagagt gagaccctgt 96840 ctcaaaaaca aaacaaaaca aaacaaaaaa agttttcagt gttgcatttg ggcaggggac 96900 tctggtattt gggaacacgt tggtgtttat aaaatagctc ttatggtagg atccttctgt 96960 agatgattac gtattttaac atcctgtctt gtatgttatt tctgacttgc tactctgtgt 97020 gcaatcactt ttattgtaaa cgtcaacatt cccatatttt cagtatcttt ctgtgatatg 97080 taatcatctg gattcttaat taatctcagt catattttgg gktttttttc ttctcttata 97140 attaaaaaaa atatatagta cttcagttcc aagaaaagat gtccatgaaa ttactagtga 97200 cactgcacca aagcctgatg ctgttttaaa gcaaggtaag gataatcttg gaataaatta 97260 caagtactgt aaaaactgct aaagtgtttt tgaagattaa atttttcttt gctctttaaa 97320 atttttcttt aaattatgtt tttatggtta tatttggttc cttgtgacct ttatttttat 97380 aaacccatgt gtcttctgtg ttataagttt ccttttcttt tttttttttg gagacagagt 97440 cttgctctgt cgcccagggt ggagtggcgc aatctcggct cactgcaacc tctgcctcct 97500 gggttcaagc aattctcttg cctcagcctc ccgagtaact gggactacag gcacacgccg 97560 ccatgcccgg ctaatttttt gttattttaa tagagctggg gtttcaccgt gttgcccagg 97620 ccggtctcga actcctgagc tcaggcagtc cacctgcctc agcctcccga agtgctagga 97680 ttacaggtgt gagccactgc gcccaaccat aggtttcctt tttttttctg tttttttttt 97740 tttttttttt tttagcttat agtcagtcat aataatctag aataatggga gagaactgtg 97800 tagtcagctg ttactataat ctagcttttg cctggtaaat aaaatattag tttctgggaa 97860 tggtagaaga aaggaatcaa agtaactttg tgttgttgtt ttttttgttt tgttttgttt 97920 tgttttgttt tgtttttttg agatggagtc ttgctctgtc gcccaggttg cagtgcagtg 97980 gtacaatctc ggtgcaccct ccacctcccg ggttcaagcg attgtcctgc ctcagtctcc 98040 caagtagctg ggactatagg ggcccaccac tatgcccggc taatttttgt gtttttagta 98100 gagacagagt ttcaccattg accaggctgg tatcaaactc ctgagctcag gcagtccacc 98160 cgccttggcc tcccaagtgc tgggattaca ggcataagcc atcacgcctg actgaggaat 98220 caaagtaatt ttggtttggt atttctcact aatgaatgta catccttaac cacaaggctc 98280 cagtagtttt atttgaggaa tatgtggtaa ttgcatctgt cacttgattt ttggcactgt 98340 aaatagttgt cttctctttg cccttattcc ttgaattcag taatatgcta tgtagattgg 98400 ttaaaatcat caagattttt tggtatataa tttattccca tatattataa aatgagaata 98460 attgtgtcta gctaatttta gttgaaggta attgttacca tagttttaat tgaagttcaa 98520 ctgaaaatgt aaaaatcatg tgtggtcagt tttcaggtat tgtatattta ttttaatggc 98580 tctgtgcctg gtattaatac tgtttgataa ttgtttgttg cataagatat tacatgattt 98640 cctgtattaa aggtttttac tagtgaagct taatctttgt ttaaaagaat aaaactgctg 98700 ttcttttatt tatttaaact taagtaatct tccaatggtg gcagttttgc catctataaa 98760 atgactacct aatgcatagg atggatagga ttaaatatat tgctacattt aaaagcactt 98820 aacacagttt ctggtacata gcaaacaata aatgtcagtg gtattagcac agtagttttt 98880 atttctttat cgtgtttaat actttttgag aaaacaattg tcttaatttc ttcagttcat 98940 gtaccttgtg gtctgtttcc agcctttgaa caggcacttg aatttcacaa aagtagaact 99000 attcctgcta actggaagac tgaattgaaa gaagatagct ctagcagtga agcagaggaa 99060 gaagaggagg aggaagatga tgaaaaagaa aaggaggrta atagcagtga agaagargta 99120 agtgaaaaca gttgatacct tttaaaatta taaataacag ttgggtttcc cttgtgggtt 99180 aggatttggc attaagtcat tactcatata agtattttta gtatgagaaa tatttcataa 99240 tcttgtattt gagatcctca taatcagata gtttacttgg ttactttaga tatatgatct 99300 tgtgaaatag aaaatattaa gcctgatttt cctaataacc tcaaaaacat gtcttagtct 99360 tcctatgctt tttttaatga gatgattaag aaagtatcca ccaggctggg cgcagtggct 99420 caggcctgta atcccagcac tttgggaggc caaggcaggc ggatcacgag gtcaggagat 99480 cgagaccatc ctggctaaca cagcgaaacc ccgtctttac taaaaataca aaaaattagc 99540 cgggggtggt ggtgggcacc tgtagtccca gctactcggt aggctgaggc aggagaatgg 99600 catgaaaccg agagacggag cttgcagtga gccaagatcg cgccactgca ctccagcctg 99660 ggcgacagag cgagactctg tctcaaaaaa aaaaaaaaac gagaacaaaa acaaaaaaac 99720 ttaccatcta ggccaggcac ggtgactcac gaccataatc gtagcacttt gggagggcaa 99780 ggcaggtggc taacttgaac tcaggagttt gagaccagac tgggcaacat ggtgaaaccc 99840 catctcaaca aaaataaaaa taaaaataat tagcagggca tgtcagtgca ctccagcccc 99900 agtgcactca gtagccccag ctactgagga ggctgtgttg ggaggacggc ctgagccctg 99960 gaggtggagg ttgcaatgag ccgtgaattt accactgccc tacatcctgg gtgacagagt 100020 gagaccctgt cttattaaaa aaaaaaaaaa aaaaaaaaaa agaacctacc tacctaattc 100080 tttaaaatgc ttttcattac catgttgaca gctgtcacct catgaaatgc ttacatctcc 100140 atattttatt gattaaccca gaaaagtata aaatacatct ttcatttaat atttaaaaga 100200 aaaatcaggc cgggtactgt ggctcatgct gggatgacta atcctagcac tctgtgaggc 100260 cgaggcgggc agatcatgag atcaggagtt cgaggccagc ttggctaaca tggtgaaacc 100320 ccgtctctta ctaaaaatac aaaataatta gccaggtgtg gtggtggaca cctgtaatcc 100380 cagctactcg ggaggctgag gcagtagaat tgcttgaacc cgggaggcgg acttgcagtg 100440 agctgagatt gcgccattgt tctccagccc gggtgacagt aggagacccc gtctcaaaaa 100500 aaaaaaaaaa aaaaatagca gaaaagtaaa gatcaatatc tctttgattc agagaatctt 100560 gaatcatttt atctaagaca aatgggtaga tagttctgat tagatcataa ataagccata 100620 aaatatggct caaagagctc aaattttgga ttttatttta cctggagctt tagatctggc 100680 agtgacaagg tactctttaa tggtttgctg aacagcctcg taaaagttaa tgagtggtgc 100740 agatagtgaa ggctgctata tttgaccatg aaatgaaatg tatttaatgt gtttagtaaa 100800 ttaattggtc agtatgtaat actttaggta agttatgaaa ttctgccttt taactgtagt 100860 aaagtattaa tgaaatgaga ttgttttgga ggttatattt tagggtgtag tgatctgtaa 100920 tgacaatgta taatgtagaa atttccctgc ttcagtcttc tccctgactg ctttctattt 100980 aaattgctaa aatcagcaat cattcccttg ctgctaaaaa cctcctgagt agctttttgt 101040 cactgtaaaa gttaagcttt catatagaat ctcctttata gtccaattcc tgcctcaatt 101100 ttttctcatc atttcccttt ctgtgtcctc ccccttgaac tttatgctat agcaatgtca 101160 aattacatat gttgctgcta ctcacctttg catatgctag tacctctgta ggcaccacct 101220 tttcctcccc tccttcctta gtcagcatgg tacattcctg ttcaattttc agaaccctac 101280 ttaggtgcca tctctctaaa gagaaacctg tgaaccatca cctccctcac ctcaggaaag 101340 caccagttca tttgctagtc taaactacta ctgctcctgg caggcacggt gggtcatgcc 101400 tgtaatccca gcactttggg aagctgacgc tagcggatca cctgaggtca ggagtttgag 101460 accagtctgg ccaacatggt gaaacccgtc tgtactaaaa atacaaaaat tagctgggtg 101520 tggctgggcg cggtggctca cgcctgtaat cccagcactt tgggaggcca aggcaggcag 101580 atcacctgag gtcaggagat cgagaccatc ctggctaaca tggtgaaacc ctgtctctac 101640 taaaaacacc aaaaaattag ccgggcatgg tggtggacgc ctgtagtccc agctactcag 101700 gaggctgagg caggagaatg gtgtcaaccc gggaggcgga gcttgcagtg agccgagatc 101760 acgccactgc actccagcct gggtgacaga gccagactcc atctaaaaaa aaaaaaatta 101820 gctgggcgtg gtggcaggtg cctgttatcc cagctactag ggaggctgag gcagcaggat 101880 tgcttgaacc caggaagcgg aggttgcagt gagccgagat gacaccactg cactccagcc 101940 tgggcgacag agcaagactc tgtctcaaaa acaagacaaa acaaaaaaag aattactact 102000 gctccttata catatttttg tcatttatat ttattgtcta ttttttcttc cccacgaatt 102060 tgtaagttcc ttaaagttag gaatgctgtc ttactcgtat tcatacttaa agtgtctgtc 102120 atagtgtgta aaatataaca gatgataaat agctaccaaa ccttttggtg cttagtgttt 102180 tcacaagtgt ttttgtaaaa gcaaatctgt agtgtagtgt aaacattttc ttgagttaaa 102240 agcttacctt aaatgttttt ggaataggaa aaattgtgct gtttattaaa gttaaatatt 102300 gtaccatggg aacatttaag ataacatcta gaaaatagat gtccttagat ttgtatttga 102360 tcaccttacc aaaacctgaa tgtatggagt gtgtttaaat tgagaaatac agattcatca 102420 ttagcaaatt aatggttgtc atttattttt ctctgcccaa ttagaaaacc taatagttga 102480 tcaacatagg ctttaaaaaa aaaattacct ttctttgttg tactagcttt taaattaaaa 102540 agacaatctc gctaaagcag tggcattagg cacatgattt tccaaattga aaaatgctat 102600 gtttttattt atttagatta ccagttgaat atcctaactt ttactgttac aaatctgtat 102660 atttaagcca gagtaactgt aaagcctagc tgtatatttg gaatcatttt ctctgcagtt 102720 actcctaatc atgttctgta acagtaggct ccatagccta ttttttcttt tatttgattc 102780 agcacctggc atagtaccta cacttacagg tgctcaagtg tttgttgaat ggatgaaagt 102840 cattgaattg gtgagttcct caatcttaac ctactaccct gctccttgca ggttgcttca 102900 ggaccagctc cctgctgcca cagcccttgg ccacagcagc aagaagctag tccttccctt 102960 gtaattgata aattggggaa ctattttatt acttgaagat ttccctaagt ctgaatcttc 103020 aggtattgta gatcaaattt gttccccact ttactgatta tgtaagcttg aagtatatac 103080 agtggggctg cataaaatga aggatacttg cagtcagcta atgggaccca tacttttata 103140 gtaccttgag gagcaataat agaattagtt ttgatgttta actctgatcc agtttcgcat 103200 aagttgagag taggtattct tgaacctgtg atcctgattt gaaaaatagc tctctcatat 103260 ggtaaaaaaa acaaaacaaa acaaaacaaa accacgaaca gtcttgctag tcccttttct 103320 catatgggaa tttttactgt ggggattcta actattggga tactttttaa ggcatattcc 103380 tctataaaac ataaaatgtc tagacttacc tggttttgaa cagcttagtg ttaaaagagt 103440 aactttgatt acgtaaaaag cctttgaagt attttaatga acactagtct ttgctattgg 103500 taagaaatct gcttgtttta ttaaaatgct taattgaaga aaataatatt cttctgtgat 103560 taaaattagg aagaaataga accatttcca gaagaaaggg agaactttct tcagcaattg 103620 tacaaattta tggaagatag aggtgagtat tttttattta tcattaacgt ggtaagtttt 103680 ggacagataa ttagtatctt aaaaataaga atagaatttt gtttactgaa ctttatgtca 103740 tcagtaatta tggctgtctt tttgactctg atatcaccag cacctagcat agcgcctgtc 103800 acgtawcaag tagaatgagg aatttgatag cattttgaca gatattgtgg ctatggacac 103860 ataaatgcac ttattaccct gtcctgccaa tccctttacc tatccatggg tccctaaaga 103920 acagtagcaa aatgggaatt aagccagaaa tagataaaga agtgtgacaa agtctcaaaa 103980 gtgagccttg gctcagctta tttaatctgc cagaagctgc taaggccacg gaaggattga 104040 aggaagaaga ataacttagg gtaatgaact aatccttccg taacctcaac actgttattc 104100 tctagcttat cttcttggtc agagtctact ttcttctttt ctgggaagat gcttgtaagg 104160 gagtaaggag aggctttcta gctggaaggg aagggcaaaa tgaatgatta gtgtttggaa 104220 acaccacaat actctgggaa ttgaacgttt tctaaatccc tgattgtaat gctgatgagt 104280 agaaactgac tctgtttgtc atgtttagtt ttcataaatg ataacaacgc ctaacacaat 104340 ataagtatat tttctttttc aatataaaat gtctttgatt cttgtattat ttctttacat 104400 gaatggaggg ttggcctaag aagtatgctg tttcataggc tcttgtataa gataaatgaa 104460 aaagcaaaga aaatgaataa aatctaatac tagaaaagaa tgaaaaaata tgattaagcc 104520 agtcatatat tttgtagaaa tggccatatg aggcattgta cccctaccct gttacctcct 104580 ctaccacttt ctcatggttt gtttgattta gcatccttgc cttgcgtttt cttgtgttag 104640 aatgaaaaaa aaaaaaaaat gtagctttac tatagaatca ttatttagag tagtagtaaa 104700 ttggtaaaat acaaagaaaa tgccataaaa gtaactctta ttgggaagca gttatctagt 104760 atttcttccc tcagcagagg gaagaaagta gaatgaaaat aaaaggtttc aagaatctgg 104820 ttatggtttt taaaatttaa aaaattttaa agtggccatg ggcttattta tgtgtagata 104880 ccatgtcttt gattaacaag ataatttctc atatatatat atacatatat atgtatatat 104940 acacacacat atatatacgt gtatatatat atatacgtgt atatatatat aaaatactat 105000 aaatacttgt attatttgaa tcttttgaaa ttttaaatta aaaatgtagt gtggatctaa 105060 tatagtttgt gttttctgta acaggtacac ctattaacaa acgacctgta cttggatatc 105120 gaaatttgaa tctctttaag ttattcagac ttgtacacaa acttggagga tttgataatg 105180 tgagtgttgt agtcaaactg aaacatcttt ttcatgtaac atgttttctc aggtatctgc 105240 catctctgaa attgtttttc taaacaacaa aacaaagttt ttcagtaaaa acctaagaag 105300 aagataaagt ggaaagtaca gctagcctcc tcagacaaaa tttaaatttt aaagattttt 105360 taggccaggc atggtggctc atgcctgcaa ccccaacact ttgggaggct gaggcgggta 105420 gatcacttta ggccaggagt tcaagaacag cctggccaac atagcaaaac cccatctcta 105480 caaagaatac aaaaatcagc cagacgtggt gacgcacgct tgtaatccca gctacatggc 105540 aggctgaggc acgagaatcg cttgaaccca ggagacagag attacagtga accaagattg 105600 caccgctgga ctgcattcca gcctgcgtga cagaatgaga ctctgtctca aaaaaaaaaa 105660 aaaaaagatt ttttttcata tcagctttct gattactgtt aaattggtca atctttcata 105720 aaccttcttt ttttatctga gtcacagcac agccatattg gtggaaatcc ttcctgtgtc 105780 tctttttcat tagacagtat gctccttaag agcagggatc tgtcttaata ttctgtatat 105840 tcttaatccc tttcagaata taaagggatt aaagcaggaa atgtttgttg aactgatctg 105900 aagaaattat tttaaggttg acgtgatgta cgatacttat ccaattgaga atattctatg 105960 gaataaaata ctttacaaat ttctgcagaa ctgcatttat tagcctgtat attttatgcg 106020 aaataataaa tcctttaaaa atgtttgtct ccatttcttt gttaaaaatt ttgatttaag 106080 agcctgaata tttttccatt ttccataact gggtttattt aaaagtcaaa tggggccagt 106140 tatggtgact catgcctgta atctcagcac tttgggaggc taaggccagg ggattgctgg 106200 agcccaggac tttgagaccc gcctgggcaa catggtgaga ctccatctcc acaaaaaaaa 106260 ttttttaatt agccaggtgt ggtggtgtgc atctatagtc ccagctactt gggttgttga 106320 gtcaggaaga tcccttgagc ccggaagttc agggctgcag tgagctgtgt ttgtgccact 106380 gttgctgcac tttagcctag gcaatagaat aagatcctgt ctccaaaaaa aaaaaaaaaa 106440 aaaaaaaaaa aaaaaaaata gtcaaatgac ttaatatttc aacatttata attttctgat 106500 atttgttaga gtactaaaac ctttattttt aaaccaaatt tagaatttat caagttttta 106560 aaaattcttt tgagtctagg accaataaat ataaaaatat actcatatat actgtgtcta 106620 tcaaaatgtt attatattaa caccttttaa tttgagagtt tttgtttcac tttgtccatt 106680 agattgaaag tggagctgtt tggaaacaag tctaccaaga tcttggaatc cctgtcttaa 106740 attcagctgc aggatacaat gttaaatgtg cttataaaaa gtaagttagt ataattgata 106800 tgattatctt cagtattttt acctagaaag caggatgaaa tttaccttat aactgaggtt 106860 aattatttct ttaaagaaat cagaacattt tactagagta gtcattctat gatagtattt 106920 tctaaagtgt attctatgat actagatcta tgagaaattc tgtgaagaaa gtttttgtgg 106980 ctaagtacat ttgtcaagta cttcatagag gttaaaaagc taatattctg aagtcaagta 107040 aatccacatt tgaatcctag ttctgccaat tattaactgt ttatgcccta gggccagtta 107100 tttattctcc taagcctcag tttcttcatt tgtaaaatgg aatgcttggc caggcacggt 107160 ggctcatgcc tgtaacttca gcactttggg aggccgaggc gggcagatca cttgaggtca 107220 ggagttcaag accagcctgg ccagcatggc gaaaccttgt ctccactaaa aatacaaaaa 107280 ttacctgagc atagggctaa cgcctgtagt cccagctact caggaggctg aggcaggaga 107340 attgcttgaa cccaggaggc ggaggttata gtgagctgag attgcgccac tgcacttcag 107400 cctgggcaac agagtaaggc tgtctcaaaa aaacaaaaaa aaacaaaaaa aaacaaaaaa 107460 aggatgctgc tgtgtgcccc agggttactg tagtgactaa atgagattta ctaaggagtc 107520 cagcctcaca cataagaaat gtgcaaaaaa tagatgctat tactaatact tttaatagct 107580 gttattgtta tgactctgag cacagtgttt taaatgaatt ttttgacaaa attttttttt 107640 tgcattgaaa agattagaaa gaactagaat aaattagaat gttaatatgg ctcaatatct 107700 gagagtttag acaaaagaaa tgaatacaca ttaaaaataa ctctatagtt atattttgaa 107760 atgttttcac caaatgtaat ttctcttcat gtttccagat acttatatgg ttttgaggag 107820 tactgtagat cagccaacat tgaatttcag atggcattgc cagagaaagt tgttaacaag 107880 caatgtaagg agtgtgaaaa tgtaaaagaa ataaaagtta aggaggaaaa tgaaacagag 107940 atcaaagaaa taaagatgga ggaggagagg aatataatac caagagaaga aaagcctatt 108000 gaggatgaaa ttgaaagaaa agaaaatatt aagccctctc tggtaaatca gatattgtta 108060 attgtctttt gctttctttg gtatattttc catatcctct ataaagttcc aaaatcaata 108120 tattgtataa tattattctt tattatttgt ttattttctt cattaagtgc tacttttgta 108180 taatttttat cataacttca aaatagctgt tagctatttt tgtctagagc tttaaagtag 108240 caggagtttc ttaaaagtaa tttataactt ttaatattag aattggttga tacctcctgt 108300 tgctaagrga taaaccatgg atataggttg aaaattttta cttttgttgg tttctttcct 108360 ctgatctttt tctagggaag taaaaagaat ttattagaat ctatacctac acattctgat 108420 caggaaaaag aagttaacat taaaaaacca gaagacaatg aaaatctgga ygacaaagat 108480 gatgacacaa ctagggtaga tgaatccctc aacataaagg tagaagctga ggaagaaaaa 108540 gcaaaatctg ggtaagaaaa ttgtttttgg ttacgatgca tttcagcttg atggattctt 108600 atcatttgct ctttgtatgt gtttatgtat gaatctaaat taattgacaa aatattaaat 108660 aatagagtga ttggcattta ttaattaaat agcattcact tctttcagag accctactga 108720 tagaaatttg tagagaaaga tcatactttg atccaccctt cttgctctca tcatggaaat 108780 tgataaaatt tttagctaga gcaaagtact ttttaggtct gctgaattac ttgataatat 108840 aaacttgggg ctatgtccat gttttatata ttgtatgttg gctatagatt ctgagagttg 108900 tatttatttc ttaagcaaaa gcaagagtgg aaaaatacta aagttttaaa aggccaaaaa 108960 ttgtatgttc tattttatgt gatagtagca tttgtttgtt ttagtcactt ggaatgatga 109020 attatttgac aatgtaattt atgcactgct agtggacccc tggattttat agataatctt 109080 tggcattgaa tcttttttct gaggcttaaa aaacataaac atatctgtta gtgtctcatt 109140 taaataagta caatgaatta aaaaaaaaat catttagcag gccaggtgcg gtggttcacg 109200 cctgtaatcc cagcactttt gggagaccgg ggcgggcaga tcacttgatg tcgggatttc 109260 aagaccagcc tggccaacat accgaaaccc tgtctcttct aaaaatacaa agaagtcagg 109320 cgtggtgaca tgtgcccata atcccagcta ctcaggaggg tgaggcagga gaatctcttg 109380 aaccctggag gcagaggttg cagtgagctg agatcgtacc actgtaggcc agcttggggg 109440 atcgagtgag actctgtctc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaatca 109500 tttagcgtac tgacatttgg ccacagtatg cagacgaaca gggaagggga aaataaaaaa 109560 tggggcttat atgaatatag taaaaaatgt ttattatact ttgttgtatt tttgtgtata 109620 aaaatatata tttgaagtat gccttactgc tgttggagac tatttcaagt attaagaaac 109680 agtaaagatt tcagcttttt gcctaaaatc ttttgattga tcagagttct gaaattttgg 109740 tggcaccata caaacatgga agaaaaacat cctgggttta gaagaatgta gtaaaagtgt 109800 ttgttgatta tactttgtta tatttctatg tgtagaagtg tatgtttaaa gtactgctaa 109860 aggagatcat tttgataaga atcttaataa agattcagct ttttaccaaa aaatctgatt 109920 tttaaatctg cagatgtttc tggaacatct gctgtttgca aggcactgtg ataaatctgt 109980 atttttttct ttgctaattt tttaagttgg tgcttctgct tattttatag aataaactgt 110040 attttgtagg tttttaacaa cccctttaac tgtataatta tatggatttt tatgtactac 110100 agatatttat tagtatatgc gtacctcaaa ctaaaatttt cctgatgtga ctgccctagt 110160 ttggtagata ttttttagaa gcattgcagt tgggactgtt tatatagtac tccctgcttt 110220 tccacagttt cagttacctg tggtcaactg tggtctgaaa acaggtgagt acagtgcaat 110280 aaaatatttt gagagagtga gacaagacca tattcacata actgttatta tagcatatta 110340 taattgcttt gttattagtc attgcttatc tcatgctgtg ccttattaat aaattaaact 110400 ttatcagagg tatgtatgta taggaaaaaa cacagtatat ataaggtttg gtactatcca 110460 cggtttcagg catccactgg ggatcttgga ctgtatccct tgcagataag aggggcttac 110520 tgtaattgat ttttgagttt tattagataa ttgcagtgga aaattttaca gtgataatta 110580 atatataggt atttttctgg catataaact ttctctaagt aataggctac taccaagaat 110640 gttcagtgtc cttcttccat tgtccagatg tgggagattg aagaagttga tatatgtaaa 110700 atgtctagtt cagtgccttg gccatagcac gtacttaata cactatcttt tttaaaattt 110760 ttgtttcatt gtaaagagtg taatatcaag tagggaagat ttgatatttc tttgtgaatt 110820 cagatgaaaa tccttgctga tacatttcgt ttcccaagat attcttctct aaaatatgac 110880 tagcttaaat gcatatgtgt gtgtatgtat gtatgtatgt atgtgagtgt atgtatgtat 110940 atgagtgcat gtatgtagtg tatgcatcaa cttaattttt tttaacctgt atgtatgttg 111000 gttgcatttt atactttcag aggctctctt tcttggcatt ctgtgttcca tctggtttag 111060 cacattagtc ttgaatttgc cactgaagtc ttagagagta gaatagtttt ctttggttga 111120 ttgccagtaa ttatgttgtt tttttcttta tacttcagtg tattattaaa gtgaatttca 111180 tttggtttgt gtctgagggt gatattaaat gtatattttt aatttttgat tgcttgattt 111240 cattaataaa agagtgttgg gtttttgttg tagattgaat gaaaatttgc ctgggctact 111300 acttaaagta tgattgtgct tgctgttggc atttagaatt gtttgtattc atcaactcag 111360 tgtattctat gatgtaagat acagcttgga gtttgtcgtc atttcaaaaa atttagaatt 111420 ttgattctat atctcatttt tatatgatga ttctgaggtg catgataaat ctcttttatt 111480 gtggccacaa taaaatatgc ttcttttccc cactatgcaa agatagagtc ttatgactta 111540 ggcaagtatt ctttcaaaaa aaaattgttt taatttacta tataaaataa aattcctcat 111600 aataatttat cattgataat cccatttctg ttaatttttt tgatatttta gccttaataa 111660 aattactttt ttataataga aatattattt tcaataacat tgttaatatt ttaatctcag 111720 gctgggtgcg gtggctcatg cctgtaatcc ctgcacttcg ggaggcccag gcaggcggat 111780 cacctgaggt caggagttcg agaccagcct ggccaacatg gtgaaacccc atctctacta 111840 aaaatacaaa aaatcagcag agcgtagtgg catgtagtcc cagcttattg ggaagctgaa 111900 gcaggagaat tggttgaacc tgggagacgg aggttgcagt gagctgagat cacgccactg 111960 tactccagcc tgggcaacag agcgagactc tgtctcaaaa aaaaagtata tatatataca 112020 cgtatatatg tgtgtgtgtg tgtgtgtgtg tatacatata tatgtatatc tcagattata 112080 gctggtttca tgaaggtttt agatttttta aatacttata tatttgactg taactgagtc 112140 tttaaaatag gaataaatct tgagaatttt tgaggaattt gtgttatagt tcagacaaaa 112200 ttgatatatt tgatactttc gtgaacacgg acttacttcc accggtccat taaacatcct 112260 gccaacactc ctgccccact atacaaaagg acaaggagaa taaaaaagca ttatagtgct 112320 gcttatatca ctatttatat attatttttg ttgcctgtat aattaattag gaatgataaa 112380 gtatattatc cctaaaaagg ttgcatatag agatataaag ctgaaatttt cagtggagga 112440 agtttttttt ttaatataaa tattatattc atcaggtggg cacagtggct cacacctata 112500 atcccagcac tttggaaggc caaagtatga agattgcttg agcccgggag ttcaagacca 112560 gcctaaacaa catagtgaga cccctgtctc tacaaaaata aaaaataaaa aactatctgg 112620 gtgtggtggt gtgtgcctgt agtcccagct acttgggatg ctgaggtggg aggatcattt 112680 gagcacagga agtcaagacc acagtgagca gtgatcaggc cacttcactc caacctggat 112740 gacacagtga gaccatgtct ccaagggggg gaaaaatata tacacacaca cacacacaca 112800 cacacacaca cacatatata tgtgtgcatg gtgtaatata tgtatatata taatcattac 112860 tcttaatata taatatagca tatattttaa tatatcatgt attttacata ttattaattt 112920 ttaaagctta aatactaaac tatctggttg ttctgcctta tgatgtttag tcatttgtga 112980 gccctaaagt gatggagtct ttaagtacag acatacagga cagcaaataa aaccagacta 113040 aatgtagcct tggatttcat attgaaattt tttaaaaaat aaatctagtg tttggaaaga 113100 tttatgattg aagattttct ccaacttgtt gaatacagat tggtggtcaa tataaaaagg 113160 atatgaatgc tcagttttag aaagtacatg gtactaagaa gataaaaatg ggtgtaaggc 113220 caggtgtggt ggctcacggc tgtaatccca gcactttggg aggccacggt gggtggatca 113280 cctgaggtca ggagtttgag accagcctga ccaacatggt gaaaccctgt cactactaaa 113340 aatacaaaaa aaattagctg ggtgtggtgg tgggtgcctg taatcccagc tacttgggag 113400 gctgaggcag gagaatcgct tgaacctggg aggcggaggt tgcagtgagc cgagattgcg 113460 ccattgcacc actccagcct gggcgacagg agtgaaaatc tatcaaaaaa aaaaaaaaaa 113520 aaaaggatgt aatactcttt ctagaatagt ttgactttta gtttcatata cttgtaattg 113580 gcctctcatc tgtctatttc tttggaacat caaagttgag aagatttgta gaagatgtta 113640 ataaatctgt ggtaatgaga gtagaaattg tgttgaagtt taaaggaaag aacaaacatg 113700 ataagatatt ggtagtacat tattagaata tttctgttta aattaaaata gtatttcagt 113760 tattataaca tttgaaaatt cggttggaga atgttttaaa gttattaaga ctgccatctg 113820 ctagtaaaaa ttattgtttc aaacttcctg aggcctccag cttgggtgac agagcaagac 113880 tctgtctcta aaaaaatgaa acaaacatct tgaggctgga aatatttgaa tgaaaaataa 113940 tttttagttg taaaatggca atgaagtata atgaattttt ttgtcaattg agatgttaaa 114000 acacttttat taataaaaca tgaaaaaatt gttagagttg accatacttg cttcttgctg 114060 ttcggcacaa attctgttct tctgactttt ggaaattttc ttttactgca cttatttttg 114120 aatcttcatt ttctctaaat gtgtctttct gtaaccttgg ttgtcttaca gttttagttt 114180 ttaatagtaa aatgaaactc tgagaatttc atacacgtgg aacctgcttt tcctggaaca 114240 atcacagcca caacttttat caatatatgt ggtttggaac tgaaaaactt atgaagtgtc 114300 ttttattaac tctgcaattt tttaaacctt tcaagagatg aaacgaataa agaagaagat 114360 gaagatgatg aagaagcaga agaggaggag gaggaggaag aagaagaaga ggatgaagat 114420 gatgatgaca acaatgagga agaggagttt gagtgctatc caccaggcat gaaagtccaa 114480 gtgcggtatg gacgagggaa aaatcaaaaa atgtatgaag ctagtattaa agattctgat 114540 gtcgaaggtg gagaggtcct ttacttggtg cattactgcg gatggaatgt gaggtaactt 114600 gagttttagc tagtgattat tacctaaaaa caattataaa atacttttct atttaaaaac 114660 ttaagtaatg gattacagat ttattagtat tctaattgag gtcatagtac cttataaaaa 114720 atagttttta gcatgtaaat acctcactta gtgacttaca attaatgttt tcactactta 114780 gaagatatcc ctaatgtcta gaaaaccgag gtctacacac ttgattttta tgtactacat 114840 gtagatcagc agacaaaaca ttgttgaaat gatcagaact tctggacatc tacaaaccaa 114900 ttatcaggct gcttgctgtc ttgcatttta aactacattg ccacagtaac ctcttttttg 114960 tattctgaag tcatttcttt ccttctgtag tcagggattt aaaatcctag gaaacattag 115020 ctattttaag aaagccttat ctccttatta acagctattg cattacagcc ttattcagaa 115080 gtctcagaaa tttattagta tctttgaaaa aatctggttg aacatgtaag cagacctgct 115140 tcactaaaat agaatgcact ttttgactgc tcctaaccta gggaactgaa aggcaagagt 115200 gaaatttggt tttaagtcat gttcaatgac ttaagcattt ttcttagata aattttcttt 115260 ggctttccaa agaaattttg tattttgatt ttatccaaaa ttggcaagcc agttttggga 115320 tctattcatt cagttatctt ttaagaatca tttattttag aatatttgat agtatatttg 115380 aaatgtagcc tataagacag atacctggaa agattaacca tagaagttta agttgtggtt 115440 tcatggtgta cctagtgtct tcaaggagga atacagttct catgctttat acttaaatta 115500 tattctgtgg tttgggaagt taataggaag gcatcatctc aaattctgtg tttgagtgtt 115560 gcaaatgaaa ttttgattct ctttaatcac ttcccacaat gccctttctc taaagaaaat 115620 cacttctgag agttttgagg ctgtccttac taacttttca ttctactttc acatacatat 115680 aaatgtactc atagaaaata tatagtattc ttacatgcat gtgttttaaa taaattatac 115740 tatacaaaat tctgtttctt aaattttttg ttgtcctgtt gtgtaatagg aagctcatga 115800 tctaatctga ttacctttct tattttgaga aaagtaccag tccgatccat tctgatgctt 115860 ggaagaaaat agcaaatagc aaatagcaac ttaagtcatg aggagatgtt cagaagtttt 115920 gttttggtct cattctttat tatgtctaat caggatacag ttttagattt taagaatacc 115980 tatcgtcaga gcttagatgt gatagtcatc cagaaacata agtttttcca gataccactt 116040 taacacactg cacaatttat cctttcacct gttggttctt tttttctgtt ttttgttttt 116100 gtttttgtct ttttttggag atagagtcta gctctgttgc ccaggctgga gtgtagtggc 116160 acgatctcta ctcactgcaa cttccacctc ccaggctcaa gcgattcttg tgctgcagcc 116220 tcctgagtag ctaggattat aggcgcccac cattgtgcct ggctaatttt cgtattttta 116280 gtagagatgg ggttttgcca tgtttgtctt gaactcctga cctcaggtga tctgccttgg 116340 cctcccaaag tgctgggatt acgggtgtca gctgttggtt cttatgctgc ctttctggta 116400 tgtgattttc ctcacctgaa ggtctggcta ttaatgaata tattccaaag aaggtacttg 116460 agatctcaga tatagctacc aaaagaatat tttgcccaat tttttacttc tagtcatttt 116520 gatcttacta aattatctta ccgaatataa atgtagaggg agttctactt ctgcctagga 116580 tataaatgta aatgtagaat atgttagttt aaaattagga agaaaaaagc tgggtgtggt 116640 gttgcatgcc tgtagtccca gctactgagg tgggaggatc acttgaaccc aggaattcga 116700 ggctgcagtg aactatgatc atgccactgt actccagcct gagtgccaga atgagacctc 116760 atctcttaaa ttaagaaata cttggattag caaaaacgaa taattactgc ataatgccca 116820 tgagatagag tcacaacacc ttttgcctct ctttttagtt tttctgatta ttttgctagt 116880 ttacatgtag aggcagtttt tcctcttggc ctttatgaag tatttgaaat tctatgaaaa 116940 agggatagaa tttaggtgtt ctccaacttt cactgtatat aatcagggct catttccttc 117000 ccttcttcct tccctccttc cctccttcct tccacggagt ttcactctta atgcccagcc 117060 tggagtgcag tggctggatc ttggctcacc gcagcctccg cctcccgggt tcaagcgctt 117120 ctcctgcctc agtctcctga gtagctgaga taggcacgca ccaccacgcc cagctaattt 117180 ttgtattttt agtagagatg gggtttctcc atgtttgtta ggctggtctc aaactcctga 117240 cctcaggtga tccacctgcc tcagcctccc aaagtgctgg gactacaggc atgagccacc 117300 gtgcccggcc aaggcttatt ttctattaat tggctcttta aacaaaaaag gcttagtaga 117360 tataagaact gagtcattca tgaaactgtt ttacaattaa gatatagctt tgttttgtta 117420 agaaaagatt gactaaaaga aagagaattc caggagtatt ttgattttag atggtagaaa 117480 gaatctctag taatttagtt tttaaatagc aggaagggaa aggaaagaat ttaaattgag 117540 gcattcaagt ttttaataat atagggagga aaatgcgacc tagttcaaca agacatgctt 117600 gtgttactgt gaacgttgcc atctatttct gagaactcaa gtgcttgtat gctatgaact 117660 ttcaaaagaa gaaaacttaa catcttgatg aagacctgta ggatggatct ctatacactc 117720 caacatcatg gagaaaacaa atgtacttca gtagattaag cagttaatct tcttagagtt 117780 aacataacta ttttatttct ttctgcaata atcagtctgt cttattgata aataagcaat 117840 aatttttatt tttgcggctc ctccagttct gaagctaatt taataaggac tgcaacaaat 117900 cctcatcagc tgtatgacaa tttttctttt atttttaggg tcttcagctt gatctctaaa 117960 tttatactta acagatgtga aattggaaat gaagatttat gcgtggaggt tcatgaaaat 118020 agttttatct tcagtggtag tcattcatga caagcagttt gggagatttg tgtggaccta 118080 tttccatggt ttttttaatc tgaaacaagt atttcagatt aaaaaaaaag agagagacag 118140 acaggacctt gttctgtcac ccaggctgga gtgcagtggt gcaatcatgg ctctccagcc 118200 tcgacttctt gggctcgagt gatcctccca cctcagcctc tggaataggt gggactctag 118260 gtgtgcatca ccacacctgg ctaatttttt atcttttgta gagatggggt ctcactatgt 118320 tgcccaagct ggtttcaaac tcctgggctc aagcagtcct cctgccttgg cttccaaaag 118380 tgctgggatt atagacatga accaatgctc ctggccatga aacatgtttt tccggtaaag 118440 agattttctc aaataatgta agagtgtctg tctttttgtt taaaaatact gtttttaggc 118500 caggcacggg tagctcatgc ccataatccc agcactttgg gaggccgagg cgggcagatc 118560 acctgaggtc aggagttcga aaccagcctg gccaacatgg cgaaacctca tctctactaa 118620 aaatacaaaa attagccaga catggtggca ggtgcctgta atccgatcta cttgggaggc 118680 tgaggcatga gaatgacttg atccaggggc acagaggttg cagtgaacca agatcacacc 118740 attgcactcc agcctgggtg acagagcaag actccatctc aaaaaaaaaa aaacaaaaca 118800 aaaaacaaaa aaatcaattt tcttgttaat ttttacattt ttcttttttt tctaactaaa 118860 atgctgtttg tttgtttgtt gtttgttttt gagatgaggt tttgctcttg ttgtctaggc 118920 tggagtgcag cgccgtgata tcatctcact gtcacctccg cctcccaggt tcaaaagatt 118980 cccctgtctc agcctcccaa gtagctggga ttgtaggcac atgccaccat gcccagcaaa 119040 tttttgtatt tttagtagag atggggtttc accatgttga ccagactggt ctcaaacgcc 119100 tgacctcagg tgatccacct ctgcccccgt aagtggtggg attataggcg tgagccactg 119160 cgcccgacct gtataaatct ttaatataat tttcatgtat gtccctattt cacagctgaa 119220 aatgttgatt tttgttatac aaaggttaac atttgggcaa gtttgttttc tactgttgtg 119280 atgaaaaaaa gtcatatttt aacatgttca caccacattt actggaaagg gcttgaactc 119340 tttaaccaca aatattataa tactcttaca gaatttttct gatgcagtga cagtatctaa 119400 aactattgtc tagcactata ttacttacta tgtcaccaag cactaggcat gtttggagta 119460 tgctcataaa ttatttttta aactcttcta aaaataacaa tttagaaaaa tacataaaag 119520 tagaaagata aaattattgc tggccggccg ggcacggtgg ctcacgcctg taatcccagc 119580 actttgggag gccgaggtgg gcggatcacc tgaggttggg agttcgagac cagttcgaga 119640 ccagcctgac caacatgaag aaaccccatc tctactaaaa aaaaacacaa aattagccag 119700 gcatggaggc acatgcctat aatcccagct actaggtagg gtgaggcagg agaatcgctc 119760 gaacctggga gacggaggtt gcggtgagcc gagattgcgc cattgcactc cagcctgggc 119820 aacaagagcg aaactccatc tcaaaaaaat agaaaaaaaa taataataaa attactaata 119880 ttatagtgtg ttctctttta gtcttttaaa atgtctccct acatttaaaa aaaatttagt 119940 tctgcttaag tagtttttaa tagtaggctc tctatttaac ttttcttagt gatgttaata 120000 ttttatagag aatcttagca taattatcta attgatccct ttcactctca agtttctaag 120060 tcgtgtcgta aatatgggat tgatttttga aagtaagggt acacattgaa attaacatgc 120120 cgtaaactag ttgagtgtgt tggatttctt tgtttagtaa caactgagtg aaactatcta 120180 gactaattgg catggaattt aaaatacatc aatattttgt aatagtgtat caatactgat 120240 tgaattttct cgctcatttc caacttggct ttctgtaatc aaagagttgc aaatttagtt 120300 agagaataat tttttgtatg ttgaagatca ccagctgcat taatgggaac ccaaaagtaa 120360 aagagacact gtaaaacttc agttgtaaaa ctcttaaata agccacaaca gttctagtgc 120420 atgttaacag ttacagagaa ctgaaaataa gctaaattat gctaaatata ttatttgaaa 120480 ttaaaaatgt gttacattta aaaaatattt tcgccaaaca aattaaatcc tcttaggagg 120540 gaatgagtta ggggatgaaa aaacctggta aaaggctttc acaaatgagt aggctcaatc 120600 ctattttttg ccatcattca gttgcttttt atcacacttt attcctttat tttccaaaca 120660 taagttgaat acccactatg tgccaaatac tgtgcttgtc accaaagata ccaagaagtg 120720 gtccttatta aggaattcat aataataata aattctttat taaggtattg atcctaccac 120780 cccttacctc ttcaccccag ttaatgcact cagtcttccc ttgatattgc ttacattccc 120840 tggcattatt attataatca cttcttttga ctcaagctgt tctttttctc tccttttgct 120900 ttatagtact catctgacaa aaccacaaga accttgatta aatctaaccc tccagctcct 120960 tgcctgcacc tgtgcagccg atcgtggttg gagtaaagca agcaaccaca atgactgttc 121020 ttattttaaa ttcattactg tgaacctcaa gtgggtcctt aattctctat gtccttcttc 121080 cattcacttt cctactttct tagaagacta tttcatactg ctgcttatgt cttcacatcc 121140 ccaacatctc ttccctatcc tcattttcag ctggtgacct tgctttgtac ctactgagag 121200 atagaaacaa gaaaaacatt tctacacact tcataagatc tgcctaccta actgcgtttg 121260 ctgccacatt ctctgtgttt tcatctttag ccatagatat acagtcatgc tactgtctga 121320 agccagtctt cataagcatc ccttcttgac tgatcaaaga catggcccat tttcttgctc 121380 tgttctgagt cattcccact acttacagat ctgtggttat ttgtcacatc tcaaggaaaa 121440 tcttttctgg agcctgactt ctactgtgac atttcttcgc tccattttca ttcagcagaa 121500 ctgtttgaaa gttacctaca cttgttactc agtttctctc ctttttctgt gtaaatccac 121560 tctcatcagc cttttgttca ctgtgtctcc acaactgtgc ttaccaggaa cactagtgac 121620 cctttgtaga taaattcctc ctgcttttct tgccctctta cgtaccttat cagtggcttt 121680 acagtcagtt gatcactcct tccgtgatat gctttgcttg ggctccagct gctccagcta 121740 caaccatttt tcttgtttat tttccttttc ttcctttctc aaaattaaga aaccactcct 121800 caattatctt ttctgtcttc ctcatcttta tgtgttttct cccttctaat ttttccccta 121860 tttctttctg atatccattt aacatttttt tggtttctgt tcctattttt ccaatcctaa 121920 cttgagtcac aattctgata ttgaattttt ctaaattatt tttccgagcc aaaggaattt 121980 gatctggaat ataactttca cctaggactc tgggtaaatt ctttcccttc aactctcctc 122040 cccctccttt tctttttttc ttcatcgttc aagctttcta cattaggact acgttatgta 122100 ttattacaaa agtgaactta attttaaaat atgaaaaaaa tttctgtttc atatctattc 122160 tcctaatatc ccttaccaca tctagaaaag tattcatttg gcatatgata agtactaaat 122220 gaatgttgaa ctaaattgga cctcctgaca acaaaatgga agtagagcag aaggtaaaat 122280 ccaaataaaa gccactggtt ttttaaagtg atacctataa taggggattg ggacagatat 122340 ggcttgaccc tctcactgga gctgcttctc acctcattta tatataactc tgtgtttcac 122400 attctaagga actgtcaaac ttttccacag agacgacacc gttttacatt ctcaccagca 122460 atgtgttagg gttccaggtt ctctgcagcc ttgtcaacac ttgttattct ctgtttatct 122520 cattgtgctt ttttgatttg cattttccta atgcaacatt ttttcatgta cttgaaatgt 122580 agagttgaac attttttcat gtacttatta gccatttgta tatcttcttt agaggaacgt 122640 cccttcaagt ttttgccatt tttgaattgg tcagttgttt tgttgttgtt gaattatagt 122700 tcttcatata ttctagatgt tattccctta tcagatacat gattgcaaat gtttcttttg 122760 ttgcctctgc ttttgatgtt atttccaaag aaacattgcc aaatccagtg ttctgaaaat 122820 tttcctttat gttttcttct aagaacttta tagttttagc tgtaatgttt acattgttgt 122880 tctaatctga gttcatattt gtatatgata tatgtgacct ctttgggtta cataaagagc 122940 tcatctctta gcaaacaaaa tggagtcgag actgagagac tggaatgaat ttgattggaa 123000 atattaattt tctgttactt tttagctatt cttagtctcc ttttgtcctt aaaaaatttt 123060 taagtcactt ttcagcttaa tttctttgtt ctttcctgct ctaaaatgtt ttgtgtcctt 123120 catttttttt ctttcttatt tccatgtaat ctgatccctg ctgtacttga gttgtgtatg 123180 ccattggatt taggaatatt gaaatagagt acaaagttgt tctactcatt ttctttgaaa 123240 taattgtcct ggtaatgata gaaagcaagg taccctggga agttgtctgc gttgtttcta 123300 tagtacagaa gtcagaggcc tataaaatga ccatttgaca gtggtttcca ttatctgtaa 123360 gtttgtatta aaagagagtg gcttttgagt taaccataga ttttgagtac aaatgaacat 123420 aaacatttcc ttcatgaaaa aaagatgata attcagttat gtgttgtact caaatgggct 123480 ttaatttttc tttgtgtatt catcttagaa tgtttactaa acttggacct gtttcattca 123540 gtagtttcgt agattggcag gtacattgat atttttgtct aagtacgcac aaataactaa 123600 ttccatacat ttatcaaata ttaacaaagt acacttttgg ggaatttaaa aaacttagca 123660 tctgcattac aatatgcaat taatggcaca gttaaccatt tgcagttgtc tttgtacgct 123720 ttggtaaaat gctgtattag tgttcttatt attacatatt acctctgcct aaagacctat 123780 acttccgttt cattttatag cgttacattg cttaggaaaa cctattgata taattactat 123840 accattccgt tgagctgttt atgtcaactc ttaaatatga ctatgctata atatttatgg 123900 ttctgtcatt ctgctgttat attcttaagt gtgaggaata ccatcgctgt ctctctgatg 123960 tagatttaag tgttatatga agtagaaata tgaaatgttt atatcatact taggaatttc 124020 attttatttt attttatttt gaagcggagt ttcgctccgt cacccaggct agagtgcagt 124080 ggcatgatct tggctcactg caacctccgc cttctgggtt caagcaattc tcctgcctca 124140 gcctcccgag tagctgggac tacaggcgtg tgccaccatg cccggctaat ttttgtattt 124200 ttaggagaga tgaggtttca ccatgctggc caggctggtc ttgaaatcct gacgtcatga 124260 tccgcctgcc ttggcctccc aaaatgttag tcttacagat gtgagccact gcacccggca 124320 ggaattttat ttttgaataa tatactactt ttttgtttca ttttattgta gatacatgat 124380 taaaagtatt aattgtgatc agttgttatt ttcatatatc ttgttttaga tagtcagatt 124440 aaaatacata taaattgaga ttatataaaa ttcataaagt aggtaaatat tgataaaatt 124500 gcttatgaag tttataatta ataatgatta acactgcttt ttatcttgac ttggtgtttt 124560 tttctattat tttgtcatac ttttatttat ttaattcagg tggggaaatc cctttccata 124620 ctaagacagt acaaatttcc actttagatg tgaggttctt aaaaactaag taagttaatt 124680 atatgacata cattaacttt tttgattata gagaggcaag atataaaata catattgtgc 124740 aacaattatt aaatgaatct attcattata gaaagcaaaa tatagatgat tttaacattt 124800 acttaatggt tggttattag atttttatct tatctataca tatttcaaag agtgaggttt 124860 caagtttata ttaatttgat tttacttacg aagttatttt taaaatacct ttattctgca 124920 tgctgtttat ttttaaatct cttaagactg tgctttttag gtatatatta actatatgct 124980 gataatattt cttttaaaat tgggtttaag aagaaccaaa agctgaattg atgtgggcat 125040 attaaccatt ttggtagtat atctggtcga ctaatgttct tttccatttt tattattaca 125100 aaatgatttc ttacataatt tgccaatgca tctgtacaaa agccataaag ttttcttttt 125160 agaaatccaa tttttctgtg gtgagagcaa gtatgctgtg gtctctgaat ggtatctttt 125220 agccttgtat gaatattttt aaacatttga aaaatcaaat atttgtgtgg tggcttcttt 125280 cagaaagttt cacgtttgta ttcacaattt gtcattactg tttagtattg tcttcttttg 125340 ttgaattgta agctcctcgc aggcaagggc tgtgtttgtt gattcattgc tgagtaccca 125400 gtgcctagca gactctgtta ttacataata gataccttag aggtgtttgt caaatgaata 125460 cgtccacatg tgtacttatg gtcttggagt ctcatataat ttttcattct acggccaggt 125520 gtggtggctc acgcctgtaa tcccagcact tcaggaggct gaggcaggca aatcacctaa 125580 ggtcaggagt tcaagacaag tctggccaac atggcaaaac ccagtctcta ctaaaaatac 125640 aaaaattagc tggacgtggt ggcacacacc tgtaatccca gctactcagg gggctgaggc 125700 aggagaatca cttgaacctg agaggcggag gttgcagtga gctgagatcg caccattgca 125760 ctccagccag ggcaacagag caagactccg tctcaaaaaa aaaaaaattt ttttttcatt 125820 gtacttcatt ttatgtaggt cagggaacat actagatctt cagaagttaa atgagtttat 125880 catgcatatg ttattttatg ttttgttagt tgaatgttaa gtgatctcaa tagaaatatt 125940 atcagacaag tcatatatat accttggaaa tattttcctt agtactttag ataaatattc 126000 aataagatac ttgtttagat tccatctgta gaattaatta cattgactgt attgataact 126060 gaattgaaaa agtataggct aatcactgtc ttaattttct aactcaatat ttgtttttaa 126120 tatttgtttc aatacttttt gtttatttca gtatttcttt ttaaatggaa gcatttagta 126180 atgctgtttc ccttgtgccc tgcaaaatta atttatttcc tgttattatg aaaaggccaa 126240 ttatgatttg taatcataat tcctaaaggt taagaccagt taaagccagg atagataaac 126300 tatcctggat tgtatatata tgtatatatt gtgtatatat atgtatatat tatatacaca 126360 tatatataaa ataacaaccc agtctacctg aattttcaca ttttcagaaa actgtattgc 126420 ttgaatctaa aattgcaaga caatttattt tattttattt tattttacat atttttttga 126480 gacagagtct tgctctgttt cccaggttgg agtgcagtag tgcagtcttg gctcactgca 126540 acctctgcct tctgggatca agtgattctc ctgcctcagc ctcctgagta gctgggatta 126600 tagacatgcg ccaccgtgcc cagctgattt ttgtattttt agtagagatg gggtttcgcc 126660 atgttagcca ggctggtctg gaagcatttt aaaaccaaaa ggtttgtatt taacttactt 126720 actaagatta ttgtattccg ccagatgcgg tggctcacac ctataatccc agcacttcgg 126780 gaggccaagg tgggcgaatc actagagccc aggagtttga gaccagcctg agaaacatag 126840 tgacaccctc tctctacaaa atataaaaaa attatccagg tgtggtggtg aatgcctgga 126900 gtcccaactg ttcaggagtc tgaggtggga caattgatac tgggaggtca aggctgcagt 126960 gagccaggat ggcgctaccg cactccagtc tgagcaacag agactctgta tgaagaagaa 127020 aaaaaaaaaa agaagattct cgtattcctt aagaatgtac tttcggggat gaatagatat 127080 cttacttgac gtggtacccc aaggagaaat tgagaaatat tttatggcct tctatttctt 127140 agctgcagtt tcactctttg ccacttcctt tccagttatt ttgttttact ttatttttat 127200 ttattttatt ttttttggag acagagtctc gctgtgtcac ccaggctgga gcacagtggt 127260 gcgatcttgg ctcactgcca cctctgcctc ctgggttcaa gtgattctcc taccccagcc 127320 tcccaagtat ctgggactac aggtgcccgc cgccacagcc agctaatttt ttttgtattt 127380 ttattagaga cggggtttca ctatgttggc caggctggtg ttgaacacct gatcttgtga 127440 tccgccctcc tcggcctccc aaagtgctag gattacaggc ataagccacc gggcctggcc 127500 tattttaatt ttttatgaaa ttcaagatat gtgaacttga gctaaaattt cccattttct 127560 atctgcattg actattttct tatctattat gtctaaggta aatttggtgt tgtctctttt 127620 taatatatac taaatgtact gtatttttaa aagtctaaat ttataaagaa gaagtaggat 127680 taactgtagc tttttgttgt agttctgaag taaattcttc tttgaagcct gtgcttcttt 127740 ggtaaggaat gagaaatctt gacccactaa tattgctttc ttaaattaat tggcaccagt 127800 aattatgcca gtctaagacg ttctttctta tataatttta atgtctgtag gaaatatggg 127860 gttttcatgt agagtttttc cccgcatgaa ttttattttt ttgagatgag tcttgctctg 127920 tcacccaggc tagggtaccg tggcatgcga tctcggctca ctgcaacctc cgcctctcgg 127980 gttcaagctc ttctcctgcc tcagcatccc aagtagctag gactacaggc gcccgctacc 128040 acgcctggct gatttttgta ttttcagtag agacacggtt tcaccatgtt ggccaggctg 128100 gtctcgaact cctgacctca tgatctgccc acctctgcct cggcctccca aagtgctggg 128160 attacaggca tgagccactg cgcccggcct aatttttatc tttttgttag agacggagtt 128220 tcaccacgtt ggccaggctg gtctcgaact cctggcctcg ggtgatctgc ctgtctgggc 128280 ctcccatagt tctgggatta caggtgtgag ccaccgtgcc cggcctcccc acatgaattt 128340 gctttttttt ttttttttcc ctattctttg aaaatatcct gtagagaaaa tagaaggaac 128400 acttgggtag tattgtaatg ggtaattaca tatacggaag gaacatttct tatttttaaa 128460 agcaatgatc agttatagtt atttaatctt ctgaccatct ttatttttct tgtaagatct 128520 atcagacaac taaagtgcta tttaacgtct cattaggttt tgaaatactt gtctgccaat 128580 atgtttgtgt cagtgatctc agcttgtatt tggtaaggac atttgtaatt cgaaaaagct 128640 tccccaacca aaattctcac atgttatttt ctccttttct tcctcttcac gccactggtc 128700 tctcactctt cactaaactt actctgaata gggcctggaa tgccccctac cgttattttc 128760 gtgttagaat cttttcccgg gcagtccatt tccttctctt ctctaattct ccacaggagt 128820 gctttttggc aagcactgtc tttcactttc attgttggaa tttcttaact tgtgaaataa 128880 aggccctgat aaatttttaa gatatgctct agttctaaaa gttattagtt ttgataacag 128940 taacaatatt gtgtaactgt gttttataca aagagcataa ttggcaaggt aaaaaaaaat 129000 gcaaaaactt agtgtcaaaa caagggcctg aattaaacaa ataaaagcac tgagaaagta 129060 gaatagaaag aagccaaata acagaagagt cataatatta gctaatattt atagatctgt 129120 taagttagat ctataaatat aatgtaatat tattatacat tatgtttgct gttagattac 129180 attattactc tatagtaagt tttcagggaa tttcttaaaa ccatacagag tattccatga 129240 gaattttttt tgttttgttt tttgagacgg agtcttgttc tgtcacccag gctggagtgc 129300 aatggcgcga tctcggctca ctgcaacctc cgcctcccgg gttgaagcga ttctccagcc 129360 tcagcctccc gagtagctag gattacaggc atgcgccatc atgcctggct aatttttgtg 129420 tttttgtaga gacagggttt caccatattg gccaggctgg tctcgaactc ctgacctcag 129480 gtgatctgtc cacctcagcc ttccaaagtg ctgggattac aggcgttagc cactgcgccc 129540 ggccccatga gaattttaaa acaaaaattt tccagaatag taaggccata ctgttaatta 129600 caactatgta tgtagtttgt ttcatctaat ttaagaattt atttttgtgt gtgtgtcctg 129660 cctgctccaa cacacttttg acatctaaac tcaactactt aacatttttc tgacatccag 129720 tcctcttgta ccatccttct tttataattg agtagaagag aacatgatta ccatatagga 129780 tttacagagc ttaggcagtg ggccccagcc tgtaccatac caagcatcca tcattttatt 129840 atttatgtgt atgttcttgc caccttgttc ttaagagaat ttaaacaaca tacattatat 129900 ctgagtaata aaggaatctt cctttatcac agtggcctta cccatgaggc agtaactgtt 129960 tttccttgtt tgatgtgttt ggtgttgcat gtaaaatata caaatgtgca atatcatgca 130020 tgtattctta aattgaaaac tactatttta ccctacttca aaatataatt gataataaaa 130080 cccaggttga attgagtcag ttttttattg atagtaaaac atgtctggta taaaactagc 130140 tacagaacag atagcctttg gttaagaaag gtagaacatg attataactt tgtttagctc 130200 acattaatgt tatgtgcagc catcatgaac tgacttcagt ttctctcata aaaaattaga 130260 aagcttgttt tgttttcttt taaaaacaaa gttgatatct tgacttcttc tttccataac 130320 tcttctaata gagcaacttc taggagttca tactgaaatc tacagaggat ggaggtgttt 130380 tcttttcgtt tcccaccatg cccccgattt tggaaggaag taaattgctt tcctaagcat 130440 tatacaattt tcgatattaa taggaaaggt aaaagtactg ctgcaaatat atggatagat 130500 gctacagtgc caggtagaag taccaaaagg agtgtattta gtagaagaat acttacttta 130560 aagcttttca aagtacttac catatatttg tacttacacc agtacttacc atatatttct 130620 acaaaaaaac ccatatatgg tatgtaccat tattttttat tttgttttta aattgtagtc 130680 ttattttata cagaaataaa ctaagcctta gattgagaat gagttgatca ggaccacata 130740 actaggaagc agggattatg ttaagtctgt cctaaagctc attccattcc ctcttattct 130800 tattacggcc ctcagcatct tccagcatat aaacagaacc ccttgcatgg ggatgaatcc 130860 tcagcttttc tggcttttac tggctacaag ccagtagttt gtaccagcag agtaactgcc 130920 caggagtttg ctacagatac tttccaggcc tatgctagag taagaatata atttattagt 130980 caagtaatga ctcaagtcat ttacttcttg atttggggac tttgttactt ccttttaata 131040 cctctaacaa tgaggacatt ttaatttaaa agctcgtttt caggtgtcca tgttctagag 131100 ttttgtgttt tctatgagta atgtatataa ctttgaacac aaaaagagaa tgtcaattag 131160 tttttaatgt ttacttcttc tagctgttat aattcttgaa tgaatgaaga acgaataaac 131220 tgtcattctt ttttttttgc tttaaacaaa tagttttatt gcagttaata gtaatttgca 131280 ccctatgaaa ttgtgaagac catcagcatt gaatcaagag gccatcacag ctgttgactc 131340 agctgtctgt acatgtaggg agcatttcag aatactgaaa aactttgcag agtaatgttt 131400 ttctggtatt ttatgagcaa gcattttttt ttttctagtt gattttgcat tatccatacg 131460 ttacaatatg gtgcttttct tttcttattt cttttttttt tttttttttg tgaaacagag 131520 tctcgctctg tcgcccaggc tggagtgcag tggcgcgatc tcagctcact gcaactccgc 131580 tcctgggttc acacgattct tctgcctcag cctcctgagt agctggggct acaggccccc 131640 accaccacgt ctggctaatt ttttgtattt ttagtagaga tggggtttca ccgtgttagc 131700 gttgatggtc ttgatctcct gacttcgtga tctgcccgcc ttggtctccc aaaagtgcta 131760 ggattactgg tgtgagccac tgtgcctggc tgtaatacgg tgcttttcta ttacacaatg 131820 ccatttaaaa ctgaagctat taccttagct ctctgatgtg tctagagaaa aattgcctac 131880 ctgctgctta agaatgaaac aaagtaaaac aaaatgtaga aaatactgtg atttatctta 131940 atacttactg tacagattca gtttctcttt gttgtagccg agtaagcagt gctagttgag 132000 gaatcatgag atcatgctct ctagtcttgg ctcggccagt agctaacttt aagtccatgg 132060 gcaagttatt acctccctct ctttgtgcct tggtagcttg ctcttcaaac caggaagctg 132120 agatgatttc caaggtcctt tcccttctaa aatgtcaaga ttctatatga gtttctcttt 132180 aatatggcat aggttatttc cgtggtatat attgtacata tttatattaa tttcctttaa 132240 ttttagatac gatgaatgga ttaaagcaga taaaatagta agacctgctg ataaaaatgt 132300 gccaaagata aaacatcgga agaaaataaa ggtaagtgct tgtttttact acacactatt 132360 agctcttaaa aagaagtttt cctccaaatg atgcaattaa atatgtgtta gtgttacact 132420 ttattgaaat agcacatttt tcaaatgcta gatttgtata taattaattt tgtcaggtta 132480 gggaaagcat tttagaaaca gtactggatg atctttgaga ctttttcagt cccaacatta 132540 tataaagcca ttcaaccaga ttttatttat ctatttctgc catttccttc ccctctagtt 132600 cccaaccagg agccataagg cagtgtgact tggagagaca taggttatta ttgtagctgt 132660 aaaccattcc aaggaagaaa aggctatctg gcaactcctt tttctcttct tctttatctg 132720 attatagtca gtggcttaga atatattcac aagatttttt tttttttttg gagacagagt 132780 ctcgctctgt tgcccaggct ggagtacagt ggtgcaacct cggctcactg caacctccac 132840 ctcccagttc aagcgattct catgcctcag cctcccgagt agctgggatt acaggcacac 132900 accaccacac ctggctaatt tttgtatttt tagtagagat ggggtttcac catgttggcc 132960 aggctggtct cgaacttctg acctcaggtg atccgcccgc cttggcctcc caaaatgccg 133020 ggattatagg tgtgagccac cacgcccagc cccacaggat cctattaacc acgaaaatgc 133080 catgaggatt tacttaggag ctcatatgga aaattaatgt tacttttatg tatgatagaa 133140 ttattcattc taagactaaa ttttcctaaa tgtttgatta gcatcttcta tgcatctgga 133200 atgatactag atatgtacca tccttaggag aattactctc actcaaaatt tatagtgctt 133260 actgctaact catgcttcag agagcttatt tttcctataa atcaacaaaa tttttaagag 133320 tagtttccag gaaatatatc ggcctttgct tatctatttg taaacaaata atttgagaaa 133380 atttctcatc ttttctcaaa acattaatat atcactgagg taggaaggtt gcccctgact 133440 tgactttaca aaagtacctg gaattttgtc attcccaacc cacatatagt gagattttaa 133500 aatgaccacg ttgcaatcgt ttcttttttt tcttgttttt ttgagacgga gtttcactct 133560 tgttgcccag gctggagtgc aatggcgcga tcttggctca ccgcaacctc catctcccag 133620 gttcaagcga ttctcctgcc tcagcctccc gagtagctgg gattacaggc atgcgccacc 133680 accccggcta cttttgtatt tttagtagag acggggtttc tccatgttgg tcaggctggt 133740 cttgacctcc tgacctcagg tgatccacct gccgcggcct cccaaagtgc tgggattaca 133800 ggtgtgagcc acctcacctg gcctcttttt ttagttagta tttaatttaa acgtattgtg 133860 attttaattc tgaagagcaa gttgtaggtt tgctagtcct aatcacctat ctgattgcta 133920 atttgattcc cgttattaaa gtataaatac aaaacctttt gatcatcctg tgtagtattt 133980 tgttaagtag ctttaaaaat ctggctgttt tgaggggctt cttatgttct ttctttttat 134040 gttaaatatt taacaacata ttctgtaata gtgagttaaa agtggacatt aacattgttt 134100 ctgataaatg ccttatgata aattacgaca tacytttttt cttaacctag aataaattag 134160 acaaagaaaa agacaaagat gaaaaatact ctccaaaaaa ctgtaaactt cggcgcttgt 134220 ccaaaccacc atttcagaca aatccatctc ctgaaatggt atccaaactg gatctcactg 134280 atgccaaaaa ctctgatact gctcatatta agtccataga aattacttcg atccttaatg 134340 gacttcaagg taaacataac aatcgttctg ttgtgcaagt atttgatttt aatttatgag 134400 tcaagttcta taaaggtaat tcagtgacat taccaactac tgttttttct accagagttt 134460 tgtttgctct cttattacag gttgaatatc tcttaactga aattcttgga accagaaatg 134520 ttttggattt cagatttttt tttgattttg gaatatttgc attccacaag ccaaatccga 134580 aaattctaaa tctgaaatgc tccagtgagc gttacctttc agaatcacgt cagcactcaa 134640 aaagtgtatg attttggagt cttcagattt tggatttggg atgttcaacc tgtacttgaa 134700 ataaaatcag ccattgattg aggcctcaaa ttttttatta ctagtagatc caagggcacc 134760 ataaaatttt ggttaacatt taatattcaa cttttagcat tgttacttta gttttttaat 134820 cttttctgtg ccatcctttc tgttgcaatg taatgcatag tttgtgttat acaacatcag 134880 accataaaag tatactgaca agttgttgta aagaacacca aactttgttt gtcctgatta 134940 tgactattat gatctctgtt tctttgagac agggtctcac tctgtagcct aggttggagt 135000 acagtggtgc aatctcagct cacttcagcc tcaacctccc agactctaaa gatcctcaca 135060 cctcagcctc ccaaggagct ggagctgcag gcgcatgcca ccatgcccgg ctaatattta 135120 tattgtttct agagacgggg ttttgccctg ttgaccaggc tcgtctcgag ctcctaggct 135180 caagcaattg gcctgtctta gcctcccgag tgctaggatt acaggcagga gccactgcac 135240 ctggactgac tgcttttttt ttttttttga gacggagtct cgctctgtca cccaggctgg 135300 agtgcagtgg cgcaatctca gctcacttgc aagctccacc tcccgggttc acactgttct 135360 cctgtctcag cttcctgagt agctgggact acaggcgccc gccactgcgc ctggctaatt 135420 ttctgtattt ttagtacaga tggggtttca ccatggtctg gatctcctga ccttatgatc 135480 cgcccacctt ggcctcccaa agtgctggga ttacaggcgt gagccaccgc gcctggcctg 135540 acatgacctc ttttatgatg ggattattgc ttagtattta aggcaaaatg aagtataggt 135600 aataaaagca atgaggattg gagcagagat ttattaaatc tgactggcag ggaaggggac 135660 atatctatga gataaaagaa tggatctcca taaaaatatc ttatcatttc tcataactta 135720 aaagaggttt catttttact tattttgaat gttaatagaa gctagttttt gtttcttatt 135780 gtagatgtgt gtttatatta actcattatt ttataaacca aatgctgtac tatattttta 135840 tttactcacc actgtactaa ttagggcaat atttttaaat tgatttttaa aaaattttcc 135900 catgtatcct acatgcaaat agtgcatgga aaatacatgt atagtttaaa gaacagtaat 135960 aatatggata ctagtatgtc tccacagtag caatcaaata ctatttactt aaaacatgta 136020 gtatgtctcc tcccagaagt aattatagtc tggacttttg tgtaatcctt gctttacttt 136080 tcttcagaat tgaattatct atatttatat tcctaagtaa tttttatttt acttcgtagt 136140 atgtctcctc ccagaagtaa ttatagtctg gacttttgtg taatccttgc tttacttttc 136200 ttcagaattg aattatctat atttatattc ctaagtaatt tttattttac ttcatttttt 136260 tagggtgttt ttttgttttg tttttgtttt tgaggcagag tctcgctctg tcacccaggc 136320 tggagtgcag tggtgctatc acgactcaag tgacctcctg ggctcaagcg acctttccat 136380 ctcagcctcc taagtagctg gaactacagg tgtgtgccac catgccaggc tattttttgt 136440 agtttttgta gagatggggt ttcaccctgt tgcccaggct tgtctcaaac tcctggactc 136500 aagcaatcca atccatctgc ctcagccttc caaagtgtta ggattatagg catgagccac 136560 tgcacccagc ctgtttttta gttttttaaa aattgagtaa caagcataag cctagtttta 136620 taagaaggct cagatgaagg ctttctgaga atgttacctg aatagagatt ctaagtcata 136680 ctctgatttc agatttagct tctctgagag tttacaaaag cccctctaga gaattcagtt 136740 ggacaggtca taagccagtt ctgataaacc taggggtcca ctacagttag aatatctgac 136800 acatttggct gggcatagta gctcactcct gtaatcccag cacttttgga ggccaaggcg 136860 ggcagattac ctgaggtcag gagtttgaga ccagcctggc caacatggtg aaaccctgtc 136920 tctactaaaa aaaaaaatac aaaaactttg ccgggtgtgg tggtgggtgc ctgtaatccc 136980 agctacttgg gaggctgagg caggagaatc gcttgaaccc aggaggcaga ggttgcagtg 137040 agtcaagatc acaccattgc actctagcct gggtagcaac agcgaaactc catcttaaaa 137100 aaaataaaat aaaaagaata tgtgacacat tcgcagtgga tgttatgtgg gagtgtgctc 137160 agtcgtagta gcagactact ctatatatat tctcattcta ggcttatggt gatttacccc 137220 tatttttcac aagtaggtca gaatgtttct gctggataaa atgatgtcat tagctgatct 137280 aggtagtggc acataagaca tggaagggag agctggaatt tcattgacac atagttgaaa 137340 caagataaat acagatttta aaatccagtc tgggtcactc aggcattcaa agactacaga 137400 gaggcggtaa ggtagcttaa tgataatagc ctgcagtatg actttctttg gaaataacac 137460 tgctgtagtc tggactggtt tccctctata tctgttgcac agctattttc ctagggagat 137520 cccattctag ggatctcctt taatgtagtt cttggatttc tttgtttttg gcatctcagg 137580 ttcctcccca cccccatact ttcattttgg tggagcacat tctctagtag cttctaaaga 137640 acaagtgcat gagatggaat ttttagagat tggctgcatt taaatatctg tatttatttt 137700 tgcaaatatc tttattctat tcatagtttg gctggattgg aagtttttcc ttcagtattt 137760 tattggcagt gttgaattgt ttttttgctt ccagtattgc ttttgtgaag tccagagctc 137820 ttccaattcc tgattctttg tatgtgactt gttttattct ttattttttg cttttctgtc 137880 tctggaatct tttttttttc ttttcttttt ttgagatgga atctcactct gttggccaag 137940 ctggagtgca gtggcacaat tttggcttac tgcagcctct gcctcctggg ttcaagtgtt 138000 tctcctgcct cagcctcccg agtaggtggg attgcagggg cctgccacca cgcctggcta 138060 gtttttctat ttttagtaga gatggggttt tgccattttg gccaggctgg tctcgaactg 138120 ctgacctcaa gtgatccact cacctcagcc tcccaaagtg ctgggattac tggcatgagc 138180 caccatgccc tgctaggaat cttgaatctt tgtttttcgt atccagaaat ttaactctga 138240 aatatatcta cacaaggcca aaggaagaga acagatatat ccacagtaca cttggctgtt 138300 ctctttcttt agccttgtgt agatatattt tcatcccctg tgatgcctac ttgctagact 138360 ctttcattct agcaacttct gtctttctgt tctaggcagt ttttttgaat gattttcttg 138420 atgatttcct ctcctgcatt ttactgttct ctccttttta atttttgttc attgttacta 138480 tttttgagat gaggtgttac tatattgccc aggctggtct caaacttctg agctcaagcg 138540 attttcctgt ctcagcctcc caaagtgcta ggattacagg catgagctac tgtcccagcc 138600 ctgccaaatt tttttttttc tttaattgag actgtgtttc actatgttca gcccaggctg 138660 ttctcacact tctgggctca agcagtcctc ctgcctcagc ctcccaagta gttgggtagc 138720 tggggtatag gtgcatgaca ctgcacttgg ctgttatctt tctttggcca gccattattt 138780 gatattatac cttctgtaat atactctaat atgctcacct tttaatttct tgctttttat 138840 taattttttt gtctttttat ttctttgaaa ttttatctgc tttgtctttt aatccttttg 138900 ttgacttttt gctgtaatgt ttaagttttc aatgattttt tttttttttt tttttttttg 138960 agactgagtc tcactctgtt gcctaggctg gagtgcagtg gcatgatctc ggctcactgc 139020 aatctctgcc ctccaagttc aagcgattct cctgcctcag cctcccgagt agctggtatt 139080 acaggcgcct gccaccgcac ccagctaatt ttttgtattt ttagtagaga tggggtttca 139140 ccatcttggc caggctggtc ttgaactcct gaccttgtga tccacctgcc tcagcctccc 139200 acagtgctgg gattataggc gtgagccact gcgcccagcc tttcaatgaa tatttttaaa 139260 ttaaactagt ttaatttttc aagagctctg ttttctgtgt gttttgtacc actctgttct 139320 tatagatgga tgcaatacct tacctttcgg attttgatct ttcaaaggat tttataattt 139380 ttggtttttt cagttacctc taatttgctt ttttctgtgt tgttgttttg agctcttccc 139440 tattaagaat tttttttcct ggctgtctta tgattatgaa ggaaagacta aactgatttg 139500 gaaattgcaa gcatatggtt ggcacttgtt gaccttgagt ttcactatcg ggtgatctgg 139560 ttggccatgt cctagggaat tcataatatg aagtctttag gtctttttct cttagactag 139620 ttggcttcca gagaaaagag ttccaatctc ttgactggag ggtggtaaga gaatggtttc 139680 cagtgttcta ggatctagtt gaagaacaga gagtggggtg gaggattctt agtggttaga 139740 tatgttcatg aatcccccta atttcagcat cgcatctgta cctgtgcctt caacagttgt 139800 tagcatctcc caggctagga gccctctcct actgtcttca gagaataaag ctctagaagg 139860 gcagtcacct tccaacctga agtgaggtgg ggatctaggc atctaagtaa tttttagtct 139920 tcacccagtg ctccttgtat gaggctcccc actgcccttt ttaattttca taggcactag 139980 ggcagtcagt aattattgag gtttctatgg taaactgggt tggtttttgg ctttcctcac 140040 tgctggtatg aggtttagcc ttctttggtg cccgtcattc atttatctgc tttctgactt 140100 gcaaattgtg ttgcttttct ttcgtatttt cccccatttt tataggttta tattatttca 140160 gaaattgcat ttatatacca tatatatgac atatgagtat atatacacac acacatacat 140220 ataacacata tacaatgacc ccaaaccccc ccacacacat atttatccta tatgtgtaat 140280 agacactcac atgtgtgcag tctttttaca gatgggatca ttaacatatt acaaactctt 140340 tttttcccct acttagtggt ataacttata aatctttcca aatcggcaca tccacatcta 140400 ctgaatagat atatcataat ttatttaatt agttgctaaa aaaagacatt ttgatccttt 140460 cccatatttt tcctgttagt tcaacaatga gtatcatatt tgcatacttt taacatttta 140520 agtattctct tcatattttc ttttttcagt gaattttatt ttttatcttt ggccacagat 140580 caaattgaag acatttcaaa tatttccagt taattaatga tcatcatatg aatgcatatg 140640 tgtatgtata tgtatacata aaaatttgat gccccttgtg agtagagaac acagacagct 140700 aagattctat cctcttgtcc tgagatgggt tgggaataaa actagaggta cagtggtagt 140760 tggagttctg tggagaaatt gaatgtctct ggagaataat cttctacatt ctggcagtgg 140820 aaatccccta gagtaaacta gatccgggat gaatcacgta aagcctatca gttaataaat 140880 cctactaaca gtagtgtcaa tcaagtttta atagggcatt cttaatcatc tcttgttaat 140940 ctcagagaac ctacagtatc tataaaacaa gaataagctg ggcacggtgg ctcacacctg 141000 taattccagc attttgggag gccaaggtag gcggatcacc tgaggtcggg agttcaagac 141060 cagcctgacc aacagggaga aacctcgtct ctactgaaaa tagaaaacag ttagccgggt 141120 gtggtggcac atgcctgtag ttccagctgc ttgggaggct gaggcaggag aatcacttga 141180 acccacgagg cggaggttgc ggtgagctga gatcacgcca ttgcgctcca gcctgggtaa 141240 caggagtgaa actccgtcta caaaaaaaaa aaaaaaaaaa agaataagat gcagtgaaaa 141300 agaaaagaat aagatgcagt gaaaaagaaa agaacaagaa agacttggag ataaaaatct 141360 aaaggccagg cacagtggct catgcctgta atcccagcac tttgggaggc caaggtgggc 141420 agatcacttg aggtcaggag ttcgagacca gcctaaccaa catggtaaaa ccgtgtctct 141480 aataaaaata ttaaaattag ccgaatgtgg tggttggtgc ctataatccc agctgctcag 141540 gaggcagaag catgagaatt gcttgaacct gcaccactgc actccagtct gggcaacaga 141600 gagagaccct gtctcaaaaa aaacaaaaac aaaaaatcta aattaaaatt cagtagagct 141660 gggcacagtg gctcacacct gtaatcccag cactttggga ggccgaggca ggtggatcat 141720 ctgaggtcag gagtttgaga ccagcctgac caatagggtg aaaccccatc tctgctaaaa 141780 atacaaaaat tagcttggcg tgtgcctgta gtcccagcta ttggggaggc cgagacagga 141840 gaattgcctg aacccgggag gtggaggttg tagtgaactg agatcttgcc agcgcactcc 141900 agcctgggca acagagcgag actctgtctc ttaaaaaaaa aaaaaaataa gcaaattcag 141960 tagagcatta agaattttaa atggaggaaa tcttcagaaa gtttaaaaga gcagtgatgg 142020 aaattttaga gaaaagatag gttacataat gtattaatct aggaaggggg aactatgaaa 142080 acagaggaga gaggccaggc gcggtggctc acgcctataa tccctgcact ttgggaggct 142140 gaggtgggcg gatcacgagg tcaggaaatc aagaccatcc tggctaacac ggtgaaaccc 142200 tgtctctact aaagtacaaa aattagctgg gtgtggtggc atgcacctgt agtcccagct 142260 actcaggagg ctgaggcagg ggaatcactt gaacctggga ggtggaggtt gcagtgaact 142320 gagattgcac cactgcactc caacctgatg acagagcaag actcagtctc aaaaaaaaaa 142380 aaagcaaata actaaagaaa aattccagaa ctgacagaca caaatcttta cattgaaatt 142440 gctcactagg taccgaataa attaatagag tcataccgta gtacatcatt atgaaattat 142500 aaaatatcaa gaatgaaaaa atcaccacaa aggaatgaga aaccaaagga aggttatggc 142560 ttataacccc ttaagcaaag aagcaataca ttcggaagga ttcagaactc tgaaggaaac 142620 aatttttaac ctagaattct gtacctcgtg aaactctcaa tcaatgaagt ttgagggtaa 142680 ggatgtattc agataagtaa ggacgatgaa tatttatatc ccatgtacct ttttaaggaa 142740 actactcgat gagctctagt acagatgaga taaccaagga agaagaagat gtgagattca 142800 gttaacagtg gacctagctc aaaagaaagc agtgaggagg attccccgga tgacagctgt 142860 tagagcagac ccagagagat accacccaga tgctactgca gaaaatagct ctctagggtg 142920 cagagatagg atgaataagg ggaaattgga tacaacgctt aatttaatga aataaaataa 142980 tgtaaaagag atacaaagga agatgtaaca tgcagaaaag tagttggaaa ctcttggaaa 143040 aataaaatgc tgtataagaa aggaagttta tcaaatgtac tacttatttc tgcagggaac 143100 cacatttaca tatgttataa atactatatt gtaaaaatga aagataacta tatatagaaa 143160 ggatggtaga ggggagatat gggtgttgat aagtaagaat ccccattgct catagaggat 143220 aaactttata aataagtcag caggccaggg acgttggctc atgcctataa tcccagtgct 143280 ttgggaggct gagatgagca gatcacttgg ggtcaagagt tcgagatcag cctgtctaaa 143340 atgatgaaac ttcatctcta ctaaaaatac aaaaattagc tgggcatggt ggcatgtgcc 143400 tgtagtccca gctgcttggg aggccgaggc aggagaattg cttgaatcca ggaagaggag 143460 gttgcagtga actgagatca caccactgta ctccagcctg ggtgacagag tgagattccg 143520 tcacacacac aaaaaaataa ataaaacaaa tagtatatta tgaatagtaa gagatgacta 143580 taactatcag atgttaacac tttggaggtg gaaaaacaga tttttatcct ctttttggtt 143640 tttcattatg agtctagatt tgtttttaat gatgtatatg tattgttttg ataatttaaa 143700 aaatgtggcc atgcagacac acattgtaaa ggtgaaatgg tttcacaatc ttagttatgt 143760 cagttatgtt ttacatttaa tgaatttagc taaagaacat gcatggattt ctcagtagag 143820 agctatagtt aaggttgtaa gttctggagt taaatgactt agatttaact accttggggg 143880 gtagattaac ctctaaaccc cagtcccttt ttttttgctt tgttttgttg tgttgctttt 143940 tgtttgtttg tttttcttat ttatttaaga ccctagtctc ttgaactata aagtggggct 144000 gataacatgt atctcgtagc attgttctga ggattgaatg agatggtctg ttcaaagtgt 144060 atcaaaagta accagcatgt agtaagttct caggaaatgt tatcttaaaa taacaataaa 144120 atgatttacc agaatgaaca cactgaagca gtaagttcca taattaattt tacataagtt 144180 atcagtaact aaaattaatt atatctattc tttaaaacat catggaaatc attgattaca 144240 aaattattgg atattcatta ttacattgaa aaatgaagct ggccaggtgt ggtggctcat 144300 gcctttaatc tcagcacttt ggaaggctga ggtaggcagg ttgctagagc tcaggagttc 144360 aagaccagcc ttggcaacat gacgagaccc tgtctctaca aaaaatacaa aaattcactg 144420 gatgtggcac acctgtagtc ccagctactt gggtggctga ggtgggagga tcatttgagc 144480 ccaggaggtt gaggctgcag tgagccatga tagtgcctgg ggaacagagt aagaccctgt 144540 ctcagaaaaa aatagagaga gagagagaaa taaagaaaga gaaaggaaag agaaaggaag 144600 ggaggcaggg agggaaagaa aataaaacaa aacaaaacaa aggaaagaaa aatgagaggc 144660 caggtgcagt agcttgctcc tgtgatccca gctactcagg aatctgaggt gggaggatct 144720 cttgagccta ggagtttgag gctgaagtgc actgtgattg tgccactgca ctccagcttg 144780 gatgagagag tgagaccgtc tctgatgaaa agaaaaatga aggctatagt ttcataagag 144840 tataaacttg gggccaggcg tggtggctca cgcctgtaat cccagcactt tgggaggccg 144900 aggagggcgg gtcacgaggt caggagatca agaccatcct ggctaacaca gtgaaacccc 144960 gtgtctacta aaaatacaaa aaattagccg ggcgtggtgg tgggcgcctg tagtcccagc 145020 tgctcaggag gctgaggcag gagaatggcg tgaacccagg aggcagagct tgctgtgagc 145080 cgacatcacg ccacttacac ttttagcttg gatgacaaag tgagactcaa aaaaaaaaaa 145140 agaagaatat taacttggag gtagcacaat ctgggattat atcctgactc cactattttc 145200 tcagatatat tatgataaaa atattatact tatataaact tggagaagct tttttctttt 145260 ttatttcagc tttattaagg tacagtttac ataaaagtga tgacttttat gtttaaaatg 145320 tgatgggttt tgacaaaagt atacagtgtt acaaccatca ccgtgatcgt aatagaacat 145380 ttccatcatt ggaagagcct cttagcctct ttgaaaccca gtttctttgc atgttacata 145440 gagataaaag ccagcaacct cgggtggctg tatacacagg acctaatata tacagtccct 145500 ggcacataga agacattgct agtgttctta tcatactcat catttttttt tctgttattt 145560 tctagcttct gaaagttctg ctgaagacag tgagcaggaa gatgagagag gtgctcaaga 145620 catggataat aatggcaaag aggaatctaa gattgatcat ttgaccaaca acagaaatga 145680 tcttatttca aaggaggaac agaacagttc atctttgcta gaagaaaaca aagttcatgc 145740 agatttggta atatccaaac cagtgtcaaa atctccagaa agattaagga aagatataga 145800 agtattatcc gaagatactg attatgaaga agatgaagtc acaaaaaaga gaaaggatgt 145860 caagaaggac acaacagata aatcttcaaa accacaaata aaacgtggta aaagaaggta 145920 ttgcaataca gaagagtgtc taaaaactgg atcacctggc aaaaaggaag agaaggccaa 145980 gaacaaagaa tcactttgca tggaaaacag tagcaacagc tcttcagatg aagatgaaga 146040 agaaacaaaa gcaaagatga caccaactaa gaaatacaat ggtttggagg aaaaaagaaa 146100 atctctacgg acaactggtt tctattcagg attttcagaa gtggcagaaa aaaggattaa 146160 acttttaaat aactctgatg aaagacttca aaacagcagg gccaaagatc gaaaagatgt 146220 ctggtcaagt attcagggac agtggcctaa aaaaacgctg aaagagcttt tttcagactc 146280 tgatactgag gctgcagctt ccccaccgca tcctgcccca gaggaggrgg tggcagagga 146340 gtcamtgcag actgtggctg aagaggagag ttgttcaccc agtgtagaac tagaaaaacc 146400 acctccagtc aatgtcgata gtaaacccat tgaagaaaaa acagtagagg tcaatgacag 146460 aaaagcagaa tttccaagta gtggcagtaa ttcagtgcta aatacccctc ctactacacc 146520 tgaatcgcct tcatcagtca ctgtaacaga aggcagccgg cagcagtctt ctgtaacagt 146580 atcagaacca ctggctccaa accaagaaga ggttcgaagt atcaagagtg aaactgatag 146640 cacaattgag gtggatagtg ttgctgggga gctccaagac ctccagtctg aagggaatag 146700 ctcgccagca ggttttgatg ccagtgtgag ctcaagcagt agtaatcagc cagaaccaga 146760 acatcctgaa aaaggtgaga aggaaaatgt gtatgttgac ttattttagg gtttcccctc 146820 ttaaagtttc aatgatttca cagtatctct tgttataacc tgaggcgatt aagtgtcata 146880 tttgtgtgaa catggtaaaa atggaaattt taaaggtaat ttgaaaatga atagtggaat 146940 gcatttaaaa gcttgagaag gctttaatgt gctttgcttg agccatccat ggcattttat 147000 tgtggaccag aacacatgct agaaattgca cccaggccca aatccaaacc tgtttgagaa 147060 ttcattatat gcagtggttg actatatggc atgagcagct taaatctatt tctgtaacat 147120 tgtttttgca attgtaatgt gcagtttctc acgaacattt tgatttattg acagacccct 147180 ccacccttaa ccaaatactg tatgtagggg tttggagcaa ccaactgtcc aggcactgct 147240 ttcctcagac aaccgtgtca aactgatttt caagcctgac taacttgtgt gagtttgtaa 147300 aggaatttga tgctttctta gatgcatagc ttccaaattg aaggaccaat gtgtacgtta 147360 ataacctaca gtaatacttt tttgattttc cgtgaaattg ttaaaagtgg aaattcaaat 147420 cggtaccttc ccaaagtatt agtgcctttc gatggtgcca tagccatact tctgtcatca 147480 tttttcttat aaaccacttc attcaggtat ctttaaatca gtatactcta ggctgacacc 147540 tggctttgga acacactttt ccatcgtaaa gacagagcac tggagtatgt tttttatata 147600 ccgtaaaaga ttttagaatg ctagctttag gttttcagca aagcttaaag agatatggtc 147660 tagatcaaat tagttaattc tatgttttct caggaatctt gacttaaaac atttctgttt 147720 taaaataaat taaaaaaata cttgcaatta aattgaaatg ttctttgctt tttttcacat 147780 ttacaagtat aactactatg attttatctg tgccatacta tctcatgcaa ctgaactatc 147840 caggcatgac taatcttcaa aatgaaagaa tcctttattt cagaatatta ggctttcaac 147900 agtaagattt tactggccag gtgtggtcac tcacacctgt aatcccagca atttgggagg 147960 tctgggaggt caaggcggtg gatcgcttga gtccaggagt tcaagaccag cctggacaac 148020 atggcaaaac cccatctcta ccaaaaatac aaacgtcagc cagctgtggt ggcgcatgcc 148080 tgtagtccca gctaccttgg gggctgatga gggagaatca cttgatcccg gaaaggctgc 148140 agtgagccaa gatcacgcca ctgcactcca gcctgggtga taaaacaaga ccctgtctca 148200 aaaaaaaaaa aaaaaaaaaa agaaaaaaca agaaaagaaa gaaaaaaaca ggttttattg 148260 ttataggttt tcttgggggt tttttttttg agatggagtc tcgctctgtt gcccaggctg 148320 gagtgcagtg gtgtgatctg ggctcactgc aacctccgtc tcctgggttc aagtgattcc 148380 cctgcctcag cctgctgagt agctgggact acaggcgtgt gccaccatgc ccagctaatt 148440 ttttgtattt ttagtagaga tggcatttcg ccatgttagc caggatggtc tctatctctt 148500 gacctcatga tctgcctgcc ttggcctccc aaagtgctag gattacaggc atgagccacc 148560 gtgcctagcc tattgttgta gtttttaaga ggttgtatcc ttattatgtt cgtaatatct 148620 tacaaaagat taaaattaac aacaaaaaaa aagaggatat cttctctgct aatagactaa 148680 gtcaacactg cccttttgaa tcttaatctt gactaggtta ataattgtgg aatttgaaag 148740 ctactcctaa atttagggta atttatatct ctttagaaat aattggtgtt tttctttttg 148800 ttgggttttt tttttttttt tttttttttt ttttgagaca tagagttttg ctcttgttgc 148860 ccaggctgga gtgcagtggt gcaatcttgg ttcactgcaa cctccacctc tcaggttcaa 148920 gagattctcc tgcctcagcc tgctgagtat ctgggattaa cgcccggcta atttttgtat 148980 tttgtttagt agagatgggg tttcgccatg ttggccaggc tggtctacga actcctgacc 149040 tcaggtaatc cacctacctt ggcctcccaa agtgctggaa ttacaggcat gagccaccgc 149100 gcccagccag aaataggttc ttaagcacct gtttcacatg gccacatttt aataaattta 149160 cttactatga atattggaga ctccccacta tatcacaagt taaaatttaa gttttactat 149220 ttagatgtag ttttttcctc ttaatttact ctacattgaa ggtttttatt tcttagtatc 149280 tgaacacttt agaattaaac tctcttggag agaaacctga caattatggt tctgtgcttc 149340 agtatggtca atatctacgt ctcctttatg tttcacttaa attgtgatat taaaatgaca 149400 ttaggtgggt cacatacttg gtgtaaaaaa taaaaaagaa atattaaaaa tttaaaaagg 149460 tattaggaaa agttgtaagt aagattatat gacccattaa aaaaaagcta ggaagttgca 149520 gacagttaat tacttgtcct gtttttgatc aaggaagtta ggttttatac acagaaggtt 149580 gatttggtgt ctgacattgg aactgaatgg agatagtact ttattagtct ctggagaaaa 149640 aatcttactt tatatagtct gagagataac atatatgaat tagacagaaa tatagcagat 149700 gttaaggacc aaatgagtag tatagaaaaa tgctacagtt cagagtagtg attgatcagt 149760 gaagcctgga acatcttagt gaagatgtag gacttgggct agtccttgaa gaaaagggag 149820 acatttattt gtaatgtttt gcaataattt cccaccaaga agatgagacg ttttttagaa 149880 ctactatgtt ggatttgtaa atggtatgca tgttaaccaa acagtgccct gggagcttag 149940 ttattcttga cataaattgg taaaaaagaa ccaagtatga attactgcta aaatttacct 150000 catttatatc atttaaaaat tatattaata tgattataag acataatgtc attaacattt 150060 taacctgtga ttaagtctta atattttggt tttattgaat cttaacaaat ttcagttttt 150120 attttcagca tatactttta attactaggc ttataaactc ccagtactat attaaggact 150180 attttcagtt tatatctgat ttttttaaag aaggatgtgc atactttgtt tgccttttta 150240 aaaaccctga cttttattat gtataagaat tgagcttcca ttaatgacag tttatttaaa 150300 aattgtagta agttctgtga caacttatra atgtcataaa gaacatgtag ttttggattg 150360 ttctatgttt ctaaaatgtg gaattaattt atacttaggg aatgttggat tttattttgt 150420 gttacttaat tcctttccct tcatagcctg tacaggtcag aaaagagtga aagatgctca 150480 gggaggagga agttcatcaa aaaagcagaa aagaagccat aaagcaacag tggtaaacaa 150540 caaaaagaag ggaaaaggca gtaagtgtga aatctctaat ttttaaaata taaaaataat 150600 agctgataat tttaccccca gtaagaaaat ggtattcagg gtatgggata gtacactatt 150660 ttgattttgt ctgtacactc aaaaaaagtc acacaaattc tgtaaggcta cttgctttaa 150720 aaaacaaaat agacaaaaaa aaaacatgca ctaggacaaa tacctaatgt aaatgatgag 150780 ttgatgggtg cagcaaacca acatggcaca tgtataccta tgtaacaaac ctgcacattg 150840 tgcacacgta ccctagaact taaagtataa taataaaaag aaaaaagaat ttctgcaaaa 150900 acaaacaaaa caaaacaaaa aaaacacagg ttttctaatc ttaggtaaat tctagtttta 150960 agcaagttgg tgttattcag cggtggtatt gattgctgat gaaaaatcaa gtaatctgtt 151020 tttgaaataa tagccaatat attataaaca tcaacatatt tgttaccttt gtattccaca 151080 gttcttttca cttgctataa aatgtatcac acattgtgaa atatttcaac tacatgttat 151140 ttttattaca gtgaaattga ttgcttagta attcttcaag gcaaaatcag catagaagta 151200 taacaatcaa gataacttta gaactataat tccaacaact ccagtctaca acatttgtct 151260 ttggtactct atattatgtg acctgagagt aactacaaac aatacttttt gctaagtatg 151320 ctccaacttt agcaacgact ccgtgcatca atccacagaa aatatatata ctgtatttct 151380 tctgagggct gcacatttta tctctctttt gtagacaaag taagaagcag aaaatatgta 151440 aagaattttt tttcaggtgc cccacactga cctcctgctg ctcttccagg agactcctgc 151500 cacctccact gcctgacctt tgctagcagc cggcttggct gcacttcagt gcagaaaagg 151560 gttattcggc cagctccccg aggttctgct gagcccatat gacttccagg cggtgaatat 151620 ggcgtccctg gggccccggg cggctgtgct cagcaaggcc atgaaagcca ggctgagaca 151680 gtccactgca tagagtgggg ccaagccttg ggtggcttag ctaggagttg ccaggggttc 151740 cagagcagca gtgggggagc tgggagggaa tttgtgtata tgtgtcaacc agtggtggag 151800 tttctttatt cccaaaattt cacagtggga ggagttgctg ctatctacct ttactatttg 151860 gaataatttc tggctttgag agtttaactc cttttttttt tttttttttt tttttttttt 151920 tggtgatgtt gttaagtgaa aaatcagtaa atatatgcca aatcagtcag tcatcaattg 151980 tttggtgtaa ctggtaggat atttaaagtg ttttttcttt cactgtgatg tttttgtctt 152040 caagagttta ctatttaaat gacatttcct aagagagcgc atcttcaatg agttatttag 152100 tatattcata taatacagag atatagtgtt ctcccatttt acttattggg attctgccat 152160 ggtaaatcag aatgagttaa ccattcaaga gaataattta gtaacatgaa ttaattctga 152220 tggaatctaa actaatactt tgtatccaga aagaggttat ctatggaaat actaatccca 152280 tcacctacct gttcacatgg taagctgtca aggtcaagta tttccatttt agtgctgaag 152340 tttaattagc aatagcagtt gtaacataat tgttacacag acttctgaat tgttcataaa 152400 atactgaata ttttattagg gtgaaactga ttattttagt caacccacag cataggagtt 152460 atttaagagt aatttcatta tacatatcaa acttcagagt ttattcccga ggagttcttt 152520 attgaatatg atgattgtgc atcattattt gtttgtggcc aaaggagcag gaaaatgttg 152580 catataccca atgttacata ttcattgtag aggtttctac aattaatcat tttaaaagat 152640 gatatatttt atgtattaac ataatagagt aaaaagccat tcagatgatt actgtatatt 152700 tgacagtcta ccaagcataa tgatagatta gtgtgagtga ttttaaaagt atacatatta 152760 ttggcagttc aggtaagaag aattttttgt ttttgctttt cctaagatgc tgctgcattt 152820 gtatatgatt ttccaggttt ctaggcaggt tgttttctgt aggactaaat tcaaatggct 152880 aattttaaat tacctactaa aatctgtgac aaatttattg ttacattttt gtttattaaa 152940 tcttttcttt ctctttcagc aaatagtagt gatagtgaag aactttcagc tggtgaaagt 153000 ataactaaga gtcagccagt caaatcagtt tccactggaa tgaagtctca tagtaccaaa 153060 tctcccgcaa ggacgcagtc tccaggaaaa tgtggaaaga atggtgataa ggatcctgat 153120 ctcaaggaac ccagtaatcg attacccaaa gtttacaaat ggagttttca gatgtgtaag 153180 tgacatgtta aattgacaag catacaaact tcatcctagt aactcttttt gttttatttt 153240 gtttttgttt ttagagacag agtctcgctc tgtcaccagg ctggagtgcg gtggtgcgat 153300 cttggctcac tgtaatcttc agcctcctgg gttcaagcca tcctcctgcc tcagcctccc 153360 aagtagctgg gattacaggc acgcgccacc acacccagct aatttttttg tgtgtttagt 153420 agagacaggg tttcaccatg ttggccagga tggtctccat ctcctgacct catgatccgc 153480 ccgcctcggc ctcccaaagt gctgggatta caggcatgag ccactgcgcc tggccttttt 153540 ttttgagaca gagtctctct gtcgcccagg ctagagtgca gtgcagtggc atagtctctg 153600 ctcactgcaa cctccatctc cctggttcaa atgattctcc tgcctcaacc tcccgagtag 153660 ttgggattac agacgcccac caccacacct ggctaatttt tttgttgttg ttgtattttt 153720 agtagacatg gggtttcacc atgttggcca ggctggtctt gaactcctga cctcaggtga 153780 tccatccgcc tctgcctcgc aaagtgctgg gattttgggc atgagccacc atgcccagcc 153840 ccaatcctag taactcttca tgccaatact ctgaaaaaga ggctttacca aacttaatag 153900 atgtactaat gtacaatgta tagaccttat ttggatcccg gtttgaataa ataaattggt 153960 taaagaaaaa ttttaaggca gttgaggcaa atgccaacac tgactagata tttctgatat 154020 ggctgggcgc agtggctcat gcctgtaatc ccagcacttt gggaggctga ggtgggtgga 154080 tcacgaggtc aggagttcga gaccagcctg accagcatgg tgaaaccctg tctctactaa 154140 taaaacaaaa aattagctgc gcgtggtggc acgcgcttgt agtcccagct actcaggagg 154200 ctgaggcagg agaattgctt gcacctggca ggtggaggtt gcagtgagcc gaggttgcgc 154260 cactgcactc cagcctgggc gacagagtga gaccccatcc cagaaaaaaa aaaaaagata 154320 cttctgatat gatgtcggta atttgattta aaagtatctg gtgttagggg tgaggaatgg 154380 ataggggtgc agatgataca ggttttgcca taaattgatg attactgaag ctggataatg 154440 agtacatggg gttcactatc cttctctcta tacttttgta tatgtgagaa atcttcataa 154500 atcttcattt aaaaaaaggt atatatatat atgttttagg tatacatata tatatatata 154560 tatatatata tatatatata tatatttttt tttttttttt tttttttttt ttaaatagag 154620 acgaggtctc attatgttgc ccaggctaat cttgaactcc tgagctcaag actgagctga 154680 tccttccacc tctacttccc aaagtgctag gattacaggt gtgagccacc acacccagcc 154740 aatatgtata tttttttaat actactctag agtttttcac acaaggaaat accttaagta 154800 ttcttaggag attgaagatt gctttagagc tttttaaaat tgccttctaa tttaaatttt 154860 tacacactct ttaaaaaaac ctaaaaaata acagagaaca gaagagaaaa atttatccac 154920 agtcttgtta ctcacaaaat gtgtacaatt tagcattttg gtgtctttcc aggttttttg 154980 ttttgttttg tttttgtgtt tttgttattt ttagacagag tctcactctg ttgcccaggc 155040 tagcgtgcag tggcacaatc tcggctcact ccaacctcca ccttccagat tcaagcgatt 155100 atcctgcccc agcctcccga gtagctggga ttgcaggcac ccgccaccat gcccagctaa 155160 tttttgtagt tttagtagag acagggtttc accatcttgg ccaggctggt atcgaactcc 155220 tgacctcgtg atccgcccac ctcggcctcc caaagtgcta ggattacagg tgtgagccac 155280 cgtgcccagc cccagtttta tttttaagtg tgatttttta ctgtggtaat actgtatatg 155340 gatgtggata tatgtagatc ttaaggtgtt taatgctgta catattcatt caacaaatac 155400 tgtgcattta ttatgtgcca ggcattgttc taggctagat aaaaaatttg gaaaacaaat 155460 atttcagaag ccttagtttt ttagttcatc tgcctcaacc ttattcagca gccatgctcg 155520 atgttctctc ctttttgtat gttaaaattt tctttaaaaa tgtctgttaa tgaaagcttt 155580 aaatttatag cggacctgga aaatatgaca agtgccgaac gcatcacaat tcttcaagaa 155640 aaacttcaag aaatcagaaa acattatctg tcattaaaat ctgaagtagc ttccattgat 155700 cggaggagaa agcgtttaaa gaagaaagag agagaaagta agtattttta ctttattttt 155760 atttatttat ttattttgag acagagtttc actcttgtcg ccaggctgga gtgcaatggc 155820 gcaatctcgg ctcactgcaa cctccacctc ctgggttcaa acagttctcc tacctcagct 155880 tcctaagtag ctggaattac aggcatgcac caccaagccc ggctaatttt gtatttttta 155940 ggtagagaca gggtttcgcc atgtcaatca ggctggtctc taactcctga cctcaggtga 156000 tctacctgtc ttggcctccc aaagtgctaa gattacaggc gtgagccaag agtgccctgc 156060 cagtattttt actttattta aacataaacc agaatttctc actctgcagt tagactgcca 156120 tgactttgtc tattttcagg caaattcttt aatttcttta tcttattttc ctcatctcta 156180 aagtgaaatt atctcaaata aaaaaattat ttcagatcat aatattcact ttcatagagt 156240 ttatactcta ccaaaaacat cctaataaga taatttcaga tattgaaagc actatgaaga 156300 aaatgatgtt aaggtagtga ttggatgggt tactttagat tacaaatggt cagtatattt 156360 ttgttgatac ctgaatgaca tgaggaagtg agataaatta aaatctggga ggaggccggg 156420 cacagtggct catgcctgta atcccagcac tttgggaggc tgcggtgggt ggattgcctg 156480 aggtcaggag gtcgagacca gcttggccaa catagtgaaa ccccgtctct actaaaaaat 156540 acaaaaaatt agctgtgcgc ggtggcgggt gcctgtaatc ccagctactt gggaggccga 156600 cgcaggagaa ttgcttaaac ccaggaggca gatgttgcag tgaacggaga tcatgccatt 156660 gcactccagc ctgggcaaca agagctaaac tctgtctcaa aaaaaaaaag acatctatcc 156720 agaaactcct tctaaacaat gttttgtaaa tatagtcacc acaaattctt tataatgaat 156780 gattttgcta aatagagcct ctctactggg ttagcattaa aagtcggttc ctaaatacta 156840 ttttaagaaa aatccatagg aaaatgctta tcctggttac caaagaaatg caaatcaaat 156900 aaggtatcat tctttttttt tggagatgga gttttgctct gtcgcccagg ctggagtgca 156960 gtggcgcgat ctcagctcac tacaacctcc gcctcctgag ttcaagcgat tatcctgcct 157020 cagtctcccg agtggctggg attacaagcg tgctgccacg cccagctaat tttttatttt 157080 tagtagagat ggggtttcac catgttggcc aggatgcagg atggctcgat ctcttgactt 157140 cgtgatccgc ctgcttcagt ctcccaaagt gctgggatta caggcatgag ccactgcgac 157200 tggcccaaat aaggtatcat tcttaccaaa aaaattaaaa ctaaaaccaa tgcaggaaca 157260 gtgtagtgaa atatataagt tatgagttgt aatatagtag aatattactc aactttgaaa 157320 agaaaaggat ctgtatgtac tgatatggaa caatctctta aaatatattg tttaaaaaaa 157380 agtcagacac tgaactatgc ttccacttgt gtgtgtgtgg tttttttttt tttttttttt 157440 ttttgagacg gagtctcggt cagtcaccag gctggagtgc agtggcgcga tcttggctca 157500 ctgcaacctc tgccttccgg ttcaagcgat tctcctgcct cagcctcccg ggtagctagg 157560 actacaggtg cgtgccgcca tgcccagcta atttttgtaa tattattatt aatttttgag 157620 acagagtatc tctctgtcat ccaggctgga gtgtagtggt gcaatcttgg ctcactgcaa 157680 ctccgcctcc cgggttcacg ccattctcct gcctcagcct ctctagtagc tgggactaca 157740 ggcgcccacc accacatctg gctaattttt tgtattttta gtagagactg agtttcattg 157800 tgttagccag gatggtctcg atctcctgac cttgtgatcc gcccacctag tcctcccaaa 157860 gtgctgggat tacaggtgtg agccaccgtg cccggcctcc acttatgttt ttaaaggtgt 157920 tgctatatct atatatttta aatttgcata tcatatctct agattctaga gataaaattc 157980 ctgtagaaca ggggttgcaa acattttctg taaagagaca ataaatatgt taggctttgt 158040 gggccatgtg gtctctgtag aaactactta tctctgccat ggtaagtgtg aaaactccca 158100 taggcaatat gtaaacaaat aggcatggct gtgttccagt acaatttttc tttccaaaga 158160 caagtaagcc agatttgccc ctggggtagt ttttttgcca gccttttttc tagagttgta 158220 atgaatatga atcaactggt agcaatgggg aagggaattt gggtgattag gaggttcctg 158280 gatgagagtg agatttgctt ttctatccta ttacccctgt actgaatgaa ttttttaaat 158340 ctgtgcatgt atttaaaaat attaatttat gaacactaat ttataacaga gaggccgggc 158400 gcaattgctc acgcctgtaa ttccagcact tagggaagcc aaggcaggca gatcacctga 158460 ggtcaggagt tcgagaccag actggccaac atgacgaaac cccatctcta ctaaaaatag 158520 aaacattagc cggacatggt ggcgcatgcc tgtaatccca gatactcagg aggctgagac 158580 aggaaaattg cttgaaccca ggaggcaaag gttgcagtga gctgagattg cactgctgca 158640 gtccagcctg ggcaacagag caagaccccc atctcaaagg aaaaaaaaaa aaaaggaggc 158700 tgaggcagga gaatggcgtg aacctggaag gcggagcttg cagtgagccg agatcgcacc 158760 actgcactcc agcctgggcg acagagtgag actccgtctc aaaaaaacaa acaggctggg 158820 cgcggtggct cacacctgta atcccggcat tttgggaggc tgagatgggc ggatcatgag 158880 gtcaggagat cgagaccatc ctggctaaca tggtgaaacc ccatctctac taaaaataca 158940 aaaaaattag ccgggcgtgg tggcgggcgc ttatagtccc agctactggg ggggctgagg 159000 caggagaatg gcgtgaactc aggaggcaga acttttagtg agccaagatc gagccactgc 159060 actccagcct gggcgacaga cagagcgaga ctttgtctca aaaaacaaac aaacaaaaaa 159120 aaacgaaaaa aggaaacagt gagagacttc ctctgatgaa aataactaat gtgttatttg 159180 ctttgtaaac ctagtggcgg agaatgcaac agttgagcta tgcactgttt tgatccaact 159240 tgaagaagca attaagcctc ccactcttgt caccatttac atgtacaaga aaactcctaa 159300 gtacagaaag atggagggat taggagggga caaatgattt ttggatggat tgcagatttt 159360 tcctgttttg atacttgttt cactgttaca aaaagtgtat tctgtatttt atttctgtgt 159420 cttgtagact aggcacagtt ctttccctct ttgacccacg caggagcctt ccctggtctg 159480 tccttttcat tacttctgta gttgggcact gtctagcttc ttgagctaca ctagcttttc 159540 ctttcttcac atcgtagaac tgtgagaatt gaccactgtt gttgtaagct aaatgatttt 159600 ggacaatata gcggaatttt agttcagagg actagtattt tctgtctatt gttagacata 159660 aatttttatt aagctcttgg tttggtctcc cttttcctta ggtgctgcta catcctcatc 159720 ctcctcttca ccttcatcca gttccataac agctgctgtt atgttaactt tagctgaacc 159780 gtcaatgtcc agcgcatcac aaaatggaat gtcagttgag tgcaggtgac agcaggactt 159840 gctaaagcac tttgcactta atggctgttg agggccactt tttttttata ctgcacagtg 159900 gcacaaaaaa atatcagaca agcactattt tatatttaaa aattgtttct tgacaagctg 159960 acttggcact taagtgcact tttttatgaa gaaaaagtac aatgaactgc ttttcctcaa 160020 gcaataattg kttccaactt gtctgggaat tgtgtgtctg gtaactggaa ggccttccac 160080 tgtggcaaat ggaggctttt cactgcctgt agagacaata cagtaagcat agttaagggg 160140 tgggtcagaa catgttaaga taacttactg tatatgtatt cccttgtatt ttgttaaagc 160200 tggaacattt gatatttttc catttattta tgaaaaaata tgaacctatt ttcatttgta 160260 caaggtaatt gttttttaaa gcaagtcacc ttagggtggc tttaattgta taagtcaagc 160320 acatgtaata aattcaaaac ctgcagttaa caggatatta gacatcaatc ctggtaacca 160380 aatattaaag attctcttta aaaaagactg aacatgttta caggtttgaa ttaggctaaa 160440 aggtcttgca gtggcttttc atggcccttc aaattggaat ggaactactg tactttgcca 160500 tttttctata aatcagtatt tttttttaat tttgatatac attgtgtgaa aaaagaaaat 160560 ggctaataaa ctgtattaaa tcttaaacaa tgtataaaga ttgtacttag ccagttcaaa 160620 gtgtatattt attcataatg aattataaca gttatatttt tgtgttttct tgtaaatgtt 160680 tcttttccct taaatacaga taattcattt gtattgctta ttttattatg agctacaaca 160740 aaaggacttc aggaacaagt aatgtattag tatggttcaa gattgttgat aggaactgtc 160800 tcaaaaggat ggtggttatt ttaaatataa atagctaatg ggggtggtag gcctataaaa 160860 ttaaatgcct tgtataaaat ccaaaatgaa tgcaaaattg ttttcacttg tattgacttt 160920 atgttgtatg attccaatct ctgttctgtt tggcacttgt atttaattct tcacctttgt 160980 aagacatttg tatattgtgg atgtgttcat tcaagctatt taatatctgg cactgttaat 161040 acacagtact ttattgtaca gactgtttta ctgttttaat tgtagttctg tgtacttttt 161100 ttggatgggg ctggcatgtt ttctttgttt cctggcaata cgacgtggga atttcaatgc 161160 gttttgttgt agatgctaac gtgtcagaat cctttacatt caacttttct aagaaaagca 161220 ttttcagtct tgtagtgtgt gcttacagta actaattttg ttgaaaatgg tttcaagtta 161280 ttcaaatttg tacaggactg taaagatttg ttgacagcaa aatgttgaag aaaaaagctt 161340 atagaataaa agctataaag tatatattag gatctgcaaa caatgaagaa ttatgtaata 161400 tattgtacaa atgtaagcaa aggctctgaa ataaaatgcc atagtttgtg aatccttgat 161460 ttttgtttct aaaagattta gtaattttag ttcatttctg tatgtgatga ctgactggaa 161520 catacatatc cagcacgtat tatcacaggg gattaattga tacacaaaaa aaggaagatt 161580 ctacctatga aaattaaaag tccattaatc agataaggaa tgtattaggc attctttttt 161640 tttttttttt ttttggacac ggagtcttgc tctgtcaccc aggctagagt gcagtggcgc 161700 aatctccagc tcactgcaac ctctatctcc cgggttcaag caattctcca gcttcagcct 161760 tccgagtagc tgggattata gacatctgcc agcactcctg gctaattttt gtatttttag 161820 cagagacggg gtttcaccgt actggtcagg ctggtctcaa actcctgacc tcatgtgatc 161880 cacccacctt ggcctcccaa tgtgctggga ttacaggcgg gagccaacac acccagccta 161940 ggcattcttt tatctttgca cacactattt tgcttgagtc tgaatttaaa tatttttctt 162000 atcacttgaa gaattgtcca aatttgaaaa ttaagtgttt tttttaaaat ttatttaaca 162060 cttgaaacca ttaccagcgg ctttttaaaa tttttaattt agttagacct ttccgggtct 162120 tttatacttc agtgtgttct attgcacatt gcaatcatct ggacattgtt aaaagtatat 162180 tcagtactca caccccactc ccaaggagtt atatttaatt ggttgggggt agtacctgga 162240 tgttgatctt taatttttaa aggtctctag tgatattaat atgcatctgg gttgagaaac 162300 actgctttgc cgcaaacttc taaaaatcta taatctagtt ttttggcccc acttattgga 162360 ctttctacca acagaaaacc tttcttggct gggcgcagtg gctcaacgcc tgtaatccta 162420 gcactttggg aggccgaggc aggcggatca 162450 2 273 DNA Homo sapiens CAAT_signal 139..147 AACCAATCC 2 ccccagagta tggactttat ttcccagaaa gccttgaggc gtaactttct gtttccatag 60 aactggtggg aaaatggcgt cgttgtttgt atccagggac caataggaac agtgtatagg 120 cgggttctaa agaactttaa ccaatccaag gtcgtctaag aggccatccg ggaaagaggt 180 aggggagggg gggaaaaaaa atctagggga ggggagaaag ggggggaacc tagagtcggt 240 gggggggaag cgatgtttgc ccgtcagtcg agt 273 3 999 DNA Homo sapiens 3 atccttgatt tttgtttcta aaagatttag taattttagt tcatttctgt atgtgatgac 60 tgactggaac atacatatcc agcacgtatt atcacagggg attaattgat acacaaaaaa 120 aggaagattc tacctatgaa aattaaaagt ccattaatca gataaggaat gtattaggca 180 ttcttttttt tttttttttt tttggacacg gagtcttgct ctgtcaccca ggctagagtg 240 cagtggcgca atctccagct cactgcaacc tctatctccc gggttcaagc aattctccag 300 cttcagcctt ccgagtagct gggattatag acatctgcca gcactcctgg ctaatttttg 360 tatttttagc agagacgggg tttcaccgta ctggtcaggc tggtctcaaa ctcctgacct 420 catgtgatcc acccaccttg gcctcccaat gtgctgggat tacaggcggg agccaacaca 480 cccagcctag gcattctttt atctttgcac acactatttt gcttgagtct gaatttaaat 540 atttttctta tcacttgaag aattgtccaa atttgaaaat taagtgtttt ttttaaaatt 600 tatttaacac ttgaaaccat taccagcggc tttttaaaat ttttaattta gttagacctt 660 tccgggtctt ttatacttca gtgtgttcta ttgcacattg caatcatctg gacattgtta 720 aaagtatatt cagtactcac accccactcc caaggagtta tatttaattg gttgggggta 780 gtacctggat gttgatcttt aatttttaaa ggtctctagt gatattaata tgcatctggg 840 ttgagaaaca ctgctttgcc gcaaacttct aaaaatctat aatctagttt tttggcccca 900 cttattggac tttctaccaa cagaaaacct ttcttggctg ggcgcagtgg ctcaacgcct 960 gtaatcctag cactttggga ggccgaggca ggcggatca 999 4 6002 DNA Homo sapiens allele 1319 5-130-257 polymorphic base A or G 4 ccggagtgag gagctcggtc gccgaagcgg agggagactc ttgagcttca tcttgccgcc 60 gccacggcca ccgcctggac ctttgcccgg agggagctgc agagggtcca tcgccgccgt 120 cctctggagg gcagcgcgat tgggggcccg gacctccagt ccggggggga tttttcgtcg 180 tccccctccc cccaaccagg gagcccgagc ggccgccaaa caaaggtacc agtcgccgcc 240 gcgggaggag gaggagccgg agcctctgcc tcagcagccg ctggacccgc cgcccttctt 300 ccccatctct cccccgggcc tgctggtttt gggggggaga aggagagagg ggactctgga 360 cgtgccaggg tcagatctcg cctccgagga aggtgcagct gaacctggtg ttttagagga 420 taccttggtc ccagagtcat c atg aag gcc ctt gat gag cct ccc tat ttg 471 Met Lys Ala Leu Asp Glu Pro Pro Tyr Leu 1 5 10 aca gtg ggc act gat gtg agt gct aaa tac aga gga gcc ttt tgt gaa 519 Thr Val Gly Thr Asp Val Ser Ala Lys Tyr Arg Gly Ala Phe Cys Glu 15 20 25 gcc aag atc aag aca gca aaa aga ctt gtc aaa gtc aag gtg aca ttt 567 Ala Lys Ile Lys Thr Ala Lys Arg Leu Val Lys Val Lys Val Thr Phe 30 35 40 aga cat gat tct tca aca gtg gaa gtt cag gat gac cac ata aag ggc 615 Arg His Asp Ser Ser Thr Val Glu Val Gln Asp Asp His Ile Lys Gly 45 50 55 cca cta aag gta gga gct att gtg gaa gtg aag aat ctt gat ggt gca 663 Pro Leu Lys Val Gly Ala Ile Val Glu Val Lys Asn Leu Asp Gly Ala 60 65 70 tat cag gaa gct gtt atc aat aaa cta aca gat gcg agt tgg tac act 711 Tyr Gln Glu Ala Val Ile Asn Lys Leu Thr Asp Ala Ser Trp Tyr Thr 75 80 85 90 gta gtt ttt gat gac gga gat gag aag aca ctg aga cga tct tca ctg 759 Val Val Phe Asp Asp Gly Asp Glu Lys Thr Leu Arg Arg Ser Ser Leu 95 100 105 tgc ctg aaa gga gag agg cat ttt gct gaa agt gaa aca tta gac cag 807 Cys Leu Lys Gly Glu Arg His Phe Ala Glu Ser Glu Thr Leu Asp Gln 110 115 120 ctc cca ctc acc aac cct gag cat ttt ggc act cca gtc ata gga aag 855 Leu Pro Leu Thr Asn Pro Glu His Phe Gly Thr Pro Val Ile Gly Lys 125 130 135 aaa aca aat aga gga aga aga tct aat cat ata cca gag gaa gag tct 903 Lys Thr Asn Arg Gly Arg Arg Ser Asn His Ile Pro Glu Glu Glu Ser 140 145 150 tca tca tcc tcc agt gat gaa gat gag gat gat agg aaa cag att gat 951 Ser Ser Ser Ser Ser Asp Glu Asp Glu Asp Asp Arg Lys Gln Ile Asp 155 160 165 170 gag cta cta ggc aaa gtt gta tgt gta gat tac att agt ttg gat aaa 999 Glu Leu Leu Gly Lys Val Val Cys Val Asp Tyr Ile Ser Leu Asp Lys 175 180 185 aag aaa gca ctg tgg ttt cct gca ttg gtg gtt tgt cct gat tgt agt 1047 Lys Lys Ala Leu Trp Phe Pro Ala Leu Val Val Cys Pro Asp Cys Ser 190 195 200 gat gag att gct gta aaa aag gac aat att ctt gtt cga tct ttc aaa 1095 Asp Glu Ile Ala Val Lys Lys Asp Asn Ile Leu Val Arg Ser Phe Lys 205 210 215 gat gga aaa ttt act tca gtt cca aga aaa gat gtc cat gaa att act 1143 Asp Gly Lys Phe Thr Ser Val Pro Arg Lys Asp Val His Glu Ile Thr 220 225 230 agt gac act gca cca aag cct gat gct gtt tta aag caa gcc ttt gaa 1191 Ser Asp Thr Ala Pro Lys Pro Asp Ala Val Leu Lys Gln Ala Phe Glu 235 240 245 250 cag gca ctt gaa ttt cac aaa agt aga act att cct gct aac tgg aag 1239 Gln Ala Leu Glu Phe His Lys Ser Arg Thr Ile Pro Ala Asn Trp Lys 255 260 265 act gaa ttg aaa gaa gat agc tct agc agt gaa gca gag gaa gaa gag 1287 Thr Glu Leu Lys Glu Asp Ser Ser Ser Ser Glu Ala Glu Glu Glu Glu 270 275 280 gag gag gaa gat gat gaa aaa gaa aag gag grt aat agc agt gaa gaa 1335 Glu Glu Glu Asp Asp Glu Lys Glu Lys Glu Xaa Asn Ser Ser Glu Glu 285 290 295 gar gaa gaa ata gaa cca ttt cca gaa gaa agg gag aac ttt ctt cag 1383 Glu Glu Glu Ile Glu Pro Phe Pro Glu Glu Arg Glu Asn Phe Leu Gln 300 305 310 caa ttg tac aaa ttt atg gaa gat aga ggt aca cct att aac aaa cga 1431 Gln Leu Tyr Lys Phe Met Glu Asp Arg Gly Thr Pro Ile Asn Lys Arg 315 320 325 330 cct gta ctt gga tat cga aat ttg aat ctc ttt aag tta ttc aga ctt 1479 Pro Val Leu Gly Tyr Arg Asn Leu Asn Leu Phe Lys Leu Phe Arg Leu 335 340 345 gta cac aaa ctt gga gga ttt gat aat att gaa agt gga gct gtt tgg 1527 Val His Lys Leu Gly Gly Phe Asp Asn Ile Glu Ser Gly Ala Val Trp 350 355 360 aaa caa gtc tac caa gat ctt gga atc cct gtc tta aat tca gct gca 1575 Lys Gln Val Tyr Gln Asp Leu Gly Ile Pro Val Leu Asn Ser Ala Ala 365 370 375 gga tac aat gtt aaa tgt gct tat aaa aaa tac tta tat ggt ttt gag 1623 Gly Tyr Asn Val Lys Cys Ala Tyr Lys Lys Tyr Leu Tyr Gly Phe Glu 380 385 390 gag tac tgt aga tca gcc aac att gaa ttt cag atg gca ttg cca gag 1671 Glu Tyr Cys Arg Ser Ala Asn Ile Glu Phe Gln Met Ala Leu Pro Glu 395 400 405 410 aaa gtt gtt aac aag caa tgt aag gag tgt gaa aat gta aaa gaa ata 1719 Lys Val Val Asn Lys Gln Cys Lys Glu Cys Glu Asn Val Lys Glu Ile 415 420 425 aaa gtt aag gag gaa aat gaa aca gag atc aaa gaa ata aag atg gag 1767 Lys Val Lys Glu Glu Asn Glu Thr Glu Ile Lys Glu Ile Lys Met Glu 430 435 440 gag gag agg aat ata ata cca aga gaa gaa aag cct att gag gat gaa 1815 Glu Glu Arg Asn Ile Ile Pro Arg Glu Glu Lys Pro Ile Glu Asp Glu 445 450 455 att gaa aga aaa gaa aat att aag ccc tct ctg gga agt aaa aag aat 1863 Ile Glu Arg Lys Glu Asn Ile Lys Pro Ser Leu Gly Ser Lys Lys Asn 460 465 470 tta tta gaa tct ata cct aca cat tct gat cag gaa aaa gaa gtt aac 1911 Leu Leu Glu Ser Ile Pro Thr His Ser Asp Gln Glu Lys Glu Val Asn 475 480 485 490 att aaa aaa cca gaa gac aat gaa aat ctg gay gac aaa gat gat gac 1959 Ile Lys Lys Pro Glu Asp Asn Glu Asn Leu Asp Asp Lys Asp Asp Asp 495 500 505 aca act agg gta gat gaa tcc ctc aac ata aag gta gaa gct gag gaa 2007 Thr Thr Arg Val Asp Glu Ser Leu Asn Ile Lys Val Glu Ala Glu Glu 510 515 520 gaa aaa gca aaa tct gga gat gaa acg aat aaa gaa gaa gat gaa gat 2055 Glu Lys Ala Lys Ser Gly Asp Glu Thr Asn Lys Glu Glu Asp Glu Asp 525 530 535 gat gaa gaa gca gaa gag gag gag gag gag gaa gaa gaa gaa gag gat 2103 Asp Glu Glu Ala Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp 540 545 550 gaa gat gat gat gac aac aat gag gaa gag gag ttt gag tgc tat cca 2151 Glu Asp Asp Asp Asp Asn Asn Glu Glu Glu Glu Phe Glu Cys Tyr Pro 555 560 565 570 cca ggc atg aaa gtc caa gtg cgg tat gga cga ggg aaa aat caa aaa 2199 Pro Gly Met Lys Val Gln Val Arg Tyr Gly Arg Gly Lys Asn Gln Lys 575 580 585 atg tat gaa gct agt att aaa gat tct gat gtt gaa ggt gga gag gtc 2247 Met Tyr Glu Ala Ser Ile Lys Asp Ser Asp Val Glu Gly Gly Glu Val 590 595 600 ctt tac ttg gtg cat tac tgc gga tgg aat gtg aga tac gat gaa tgg 2295 Leu Tyr Leu Val His Tyr Cys Gly Trp Asn Val Arg Tyr Asp Glu Trp 605 610 615 att aaa gca gat aaa ata gta aga cct gct gat aaa aat gtg cca aag 2343 Ile Lys Ala Asp Lys Ile Val Arg Pro Ala Asp Lys Asn Val Pro Lys 620 625 630 ata aaa cat cgg aag aaa ata aag aat aaa tta gac aaa gaa aaa gac 2391 Ile Lys His Arg Lys Lys Ile Lys Asn Lys Leu Asp Lys Glu Lys Asp 635 640 645 650 aaa gat gaa aaa tac tct cca aaa aac tgt aaa ctt cgg cgc ttg tcc 2439 Lys Asp Glu Lys Tyr Ser Pro Lys Asn Cys Lys Leu Arg Arg Leu Ser 655 660 665 aaa cca cca ttt cag aca aat cca tct cct gaa atg gta tcc aaa ctg 2487 Lys Pro Pro Phe Gln Thr Asn Pro Ser Pro Glu Met Val Ser Lys Leu 670 675 680 gat ctc act gat gcc aaa aac tct gat act gct cat att aag tcc ata 2535 Asp Leu Thr Asp Ala Lys Asn Ser Asp Thr Ala His Ile Lys Ser Ile 685 690 695 gaa att act tcg atc ctt aat gga ctt caa gct tct gaa agt tct gct 2583 Glu Ile Thr Ser Ile Leu Asn Gly Leu Gln Ala Ser Glu Ser Ser Ala 700 705 710 gaa gac agt gag cag gaa gat gag aga ggt gct caa gac atg gat aat 2631 Glu Asp Ser Glu Gln Glu Asp Glu Arg Gly Ala Gln Asp Met Asp Asn 715 720 725 730 aat ggc aaa gag gaa tct aag att gat cat ttg acc aac aac aga aat 2679 Asn Gly Lys Glu Glu Ser Lys Ile Asp His Leu Thr Asn Asn Arg Asn 735 740 745 gat ctt att tca aag gag gaa cag aac agt tca tct ttg cta gaa gaa 2727 Asp Leu Ile Ser Lys Glu Glu Gln Asn Ser Ser Ser Leu Leu Glu Glu 750 755 760 aac aaa gtt cat gca gat ttg gta ata tcc aaa cca gtg tca aaa tct 2775 Asn Lys Val His Ala Asp Leu Val Ile Ser Lys Pro Val Ser Lys Ser 765 770 775 cca gaa aga tta agg aaa gat ata gaa gta tta tcc gaa gat act gat 2823 Pro Glu Arg Leu Arg Lys Asp Ile Glu Val Leu Ser Glu Asp Thr Asp 780 785 790 tat gaa gaa gat gaa gtc aca aaa aag aga aag gat gtc aag aag gac 2871 Tyr Glu Glu Asp Glu Val Thr Lys Lys Arg Lys Asp Val Lys Lys Asp 795 800 805 810 aca aca gat aaa tct tca aaa cca caa ata aaa cgt ggt aaa aga agg 2919 Thr Thr Asp Lys Ser Ser Lys Pro Gln Ile Lys Arg Gly Lys Arg Arg 815 820 825 tat tgc aat aca gaa gag tgt cta aaa act gga tca cct ggc aaa aag 2967 Tyr Cys Asn Thr Glu Glu Cys Leu Lys Thr Gly Ser Pro Gly Lys Lys 830 835 840 gaa gag aag gcc aag aac aaa gaa tca ctt tgc atg gaa aac agt agc 3015 Glu Glu Lys Ala Lys Asn Lys Glu Ser Leu Cys Met Glu Asn Ser Ser 845 850 855 aac agc tct tca gat gaa gat gaa gaa gaa aca aaa gca aag atg aca 3063 Asn Ser Ser Ser Asp Glu Asp Glu Glu Glu Thr Lys Ala Lys Met Thr 860 865 870 cca act aag aaa tac aat ggt ttg gag gaa aaa aga aaa tct cta cgg 3111 Pro Thr Lys Lys Tyr Asn Gly Leu Glu Glu Lys Arg Lys Ser Leu Arg 875 880 885 890 aca act ggt ttc tat tca gga ttt tca gaa gtg gca gaa aaa agg att 3159 Thr Thr Gly Phe Tyr Ser Gly Phe Ser Glu Val Ala Glu Lys Arg Ile 895 900 905 aaa ctt tta aat aac tct gat gaa aga ctt caa aac agc agg gcc aaa 3207 Lys Leu Leu Asn Asn Ser Asp Glu Arg Leu Gln Asn Ser Arg Ala Lys 910 915 920 gat cga aaa gat gtc tgg tca agt att cag gga cag tgg cct aaa aaa 3255 Asp Arg Lys Asp Val Trp Ser Ser Ile Gln Gly Gln Trp Pro Lys Lys 925 930 935 acg ctg aaa gag ctt ttt tca gac tct gat act gag gct gca gct tcc 3303 Thr Leu Lys Glu Leu Phe Ser Asp Ser Asp Thr Glu Ala Ala Ala Ser 940 945 950 cca ccg cat cct gcc cca gag gag grg gtg gca gag gag tca mtg cag 3351 Pro Pro His Pro Ala Pro Glu Glu Xaa Val Ala Glu Glu Ser Xaa Gln 955 960 965 970 act gtg gct gaa gag gag agt tgt tca ccc agt gta gaa cta gaa aaa 3399 Thr Val Ala Glu Glu Glu Ser Cys Ser Pro Ser Val Glu Leu Glu Lys 975 980 985 cca cct cca gtc aat gtc gat agt aaa ccc att gaa gaa aaa aca gta 3447 Pro Pro Pro Val Asn Val Asp Ser Lys Pro Ile Glu Glu Lys Thr Val 990 995 1000 gag gtc aat gac aga aaa gca gaa ttt cca agt agt ggc agt aat tca 3495 Glu Val Asn Asp Arg Lys Ala Glu Phe Pro Ser Ser Gly Ser Asn Ser 1005 1010 1015 gtg cta aat acc cct cct act aca cct gaa tcg cct tca tca gtc act 3543 Val Leu Asn Thr Pro Pro Thr Thr Pro Glu Ser Pro Ser Ser Val Thr 1020 1025 1030 gta aca gaa ggc agc cgg cag cag tct tct gta aca gta tca gaa cca 3591 Val Thr Glu Gly Ser Arg Gln Gln Ser Ser Val Thr Val Ser Glu Pro 1035 1040 1045 1050 ctg gct cca aac caa gaa gag gtt cga agt atc aag agt gaa act gat 3639 Leu Ala Pro Asn Gln Glu Glu Val Arg Ser Ile Lys Ser Glu Thr Asp 1055 1060 1065 agc aca att gag gtg gat agt gtt gct ggg gag ctc caa gac ctc cag 3687 Ser Thr Ile Glu Val Asp Ser Val Ala Gly Glu Leu Gln Asp Leu Gln 1070 1075 1080 tct gaa ggg aat agc tcg cca gca ggt ttt gat gcc agt gtg agc tca 3735 Ser Glu Gly Asn Ser Ser Pro Ala Gly Phe Asp Ala Ser Val Ser Ser 1085 1090 1095 agc agt agt aat cag cca gaa cca gaa cat cct gaa aaa gcc tgt aca 3783 Ser Ser Ser Asn Gln Pro Glu Pro Glu His Pro Glu Lys Ala Cys Thr 1100 1105 1110 ggt cag aaa aga gtg aaa gat gct cag gga gga gga agt tca tca aaa 3831 Gly Gln Lys Arg Val Lys Asp Ala Gln Gly Gly Gly Ser Ser Ser Lys 1115 1120 1125 1130 aag cag aaa aga agc cat aaa gca aca gtg gta aac aac aaa aag aag 3879 Lys Gln Lys Arg Ser His Lys Ala Thr Val Val Asn Asn Lys Lys Lys 1135 1140 1145 gga aaa ggc aca aat agt agt gat agt gaa gaa ctt tca gct ggt gaa 3927 Gly Lys Gly Thr Asn Ser Ser Asp Ser Glu Glu Leu Ser Ala Gly Glu 1150 1155 1160 agt ata act aag agt cag cca gtc aaa tca gtt tcc act gga atg aag 3975 Ser Ile Thr Lys Ser Gln Pro Val Lys Ser Val Ser Thr Gly Met Lys 1165 1170 1175 tct cat agt acc aaa tct ccc gca agg acg cag tct cca gga aaa tgt 4023 Ser His Ser Thr Lys Ser Pro Ala Arg Thr Gln Ser Pro Gly Lys Cys 1180 1185 1190 gga aag aat ggt gat aag gat cct gat ctc aag gaa ccc agt aat cga 4071 Gly Lys Asn Gly Asp Lys Asp Pro Asp Leu Lys Glu Pro Ser Asn Arg 1195 1200 1205 1210 tta ccc aaa gtt tac aaa tgg agt ttt cag atg tcg gac ctg gaa aat 4119 Leu Pro Lys Val Tyr Lys Trp Ser Phe Gln Met Ser Asp Leu Glu Asn 1215 1220 1225 atg aca agt gcc gaa cgc atc aca att ctt caa gaa aaa ctt caa gaa 4167 Met Thr Ser Ala Glu Arg Ile Thr Ile Leu Gln Glu Lys Leu Gln Glu 1230 1235 1240 atc aga aaa cat tat ctg tca tta aaa tct gaa gta gct tcc att gat 4215 Ile Arg Lys His Tyr Leu Ser Leu Lys Ser Glu Val Ala Ser Ile Asp 1245 1250 1255 cgg agg aga aag cgt tta aag aag aaa gag aga gaa agt gct gct aca 4263 Arg Arg Arg Lys Arg Leu Lys Lys Lys Glu Arg Glu Ser Ala Ala Thr 1260 1265 1270 tcc tca tcc tcc tct tca cct tca tcc agt tcc ata aca gct gct gtt 4311 Ser Ser Ser Ser Ser Ser Pro Ser Ser Ser Ser Ile Thr Ala Ala Val 1275 1280 1285 1290 atg tta act tta gct gaa ccg tca atg tcc agc gca tca caa aat gga 4359 Met Leu Thr Leu Ala Glu Pro Ser Met Ser Ser Ala Ser Gln Asn Gly 1295 1300 1305 atg tca gtt gag tgc agg tga cagcaggact tgctaaagca ctttgcactt 4410 Met Ser Val Glu Cys Arg 1310 aatggctgtt gagggccact ttttttttat actgcacagt ggcacaaaaa aatatcagac 4470 aagcactatt ttatatttaa aaattgtttc ttgacaagct gacttggcac ttaagtgcac 4530 ttttttatga agaaaaagta caatgaactg cttttcctca agcaataatt gkttccaact 4590 tgtctgggaa ttgtgtgtct ggtaactgga aggccttcca ctgtggcaaa tggaggcttt 4650 tcactgcctg tagagacaat acagtaagca tagttaaggg gtgggtcaga acatgttaag 4710 ataacttact gtatatgtat tcccttgtat tttgttaaag ctggaacatt tgatattttt 4770 ccatttattt atgaaaaaat atgaacctat tttcatttgt acaaggtaat tgttttttaa 4830 agcaagtcac cttagggtgg ctttaattgt ataagtcaag cacatgtaat aaattcaaaa 4890 cctgcagtta acaggatatt agacatcaat cctggtaacc aaatattaaa gattctcttt 4950 aaaaaagact gaacatgttt acaggtttga attaggctaa aaggtcttgc agtggctttt 5010 catggccctt caaattggaa tggaactact gtactttgcc atttttctat aaatcagtat 5070 ttttttttaa ttttgatata cattgtgtga aaaaagaaaa tggctaataa actgtattaa 5130 atcttaaaca atgtataaag attgtactta gccagttcaa agtgtatatt tattcataat 5190 gaattataac agttatattt ttgtgttttc ttgtaaatgt ttcttttccc ttaaatacag 5250 ataattcatt tgtattgctt attttattat gagctacaac aaaaggactt caggaacaag 5310 taatgtatta gtatggttca agattgttga taggaactgt ctcaaaagga tggtggttat 5370 tttaaatata aatagctaat gggggtggta ggcctataaa attaaatgcc ttgtataaaa 5430 tccaaaatga atgcaaaatt gttttcactt gtattgactt tatgttgtat gattccaatc 5490 tctgttctgt ttggcacttg tatttaattc ttcacctttg taagacattt gtatattgtg 5550 gatgtgttca ttcaagctat ttaatatctg gcactgttaa tacacagtac tttattgtac 5610 agactgtttt actgttttaa ttgtagttct gtgtactttt tttggatggg gctggcatgt 5670 tttctttgtt tcctggcaat acgacgtggg aatttcaatg cgttttgttg tagatgctaa 5730 cgtgtcagaa tcctttacat tcaacttttc taagaaaagc attttcagtc ttgtagtgtg 5790 tgcttacagt aactaatttt gttgaaaatg gtttcaagtt attcaaattt gtacaggact 5850 gtaaagattt gttgacagca aaatgttgaa gaaaaaagct tatagaataa aagctataaa 5910 gtatatatta ggatctgcaa acaatgaaga attatgtaat atattgtaca aatgtaagca 5970 aaggctctga aataaaatgc catagtttgt ga 6002 5 392 DNA Homo sapiens 5 ccggagtgag gagctcggtc gccgaagcgg agggagactc ttgagcttca tcttgccgcc 60 gccacggcca ccgcctggac ctttgcccgg agggagctgc agagggtcca tcgccgccgt 120 cctctggagg gcagcgcgat tgggggcccg gacctccagt ccggggggga tttttcgtcg 180 tccccctccc cccaaccagg gagcccgagc ggccgccaaa caaaggtacc agtcgccgcc 240 gcgggaggag gaggagccgg agcctctgcc tcagcagccg ctggacccgc cgcccttctt 300 ccccatctct cccccgggcc tgctggtttt gggggggaga aggagagagg ggactctgga 360 cgtgccaggg tcagatctcg cctccgagga ag 392 6 55 DNA Homo sapiens 6 gtgcagctga acctggtgtt ttagaggata ccttggtccc agagtcatca tgaag 55 7 111 DNA Homo sapiens 7 gcccttgatg agcctcccta tttgacagtg ggcactgatg tgagtgctaa atacagagga 60 gccttttgtg aagccaagat caagacagca aaaagacttg tcaaagtcaa g 111 8 66 DNA Homo sapiens 8 gtgacattta gacatgattc ttcaacagtg gaagttcagg atgaccacat aaagggccca 60 ctaaag 66 9 91 DNA Homo sapiens 9 gtaggagcta ttgtggaagt gaagaatctt gatggtgcat atcaggaagc tgttatcaat 60 aaactaacag atgcgagttg gtacactgta g 91 10 80 DNA Homo sapiens 10 tttttgatga cggagatgag aagacactga gacgatcttc actgtgcctg aaaggagaga 60 ggcattttgc tgaaagtgaa 80 11 92 DNA Homo sapiens 11 acattagacc agctcccact caccaaccct gagcattttg gcactccagt cataggaaag 60 aaaacaaata gaggaagaag atctaatcat at 92 12 139 DNA Homo sapiens 12 accagaggaa gagtcttcat catcctccag tgatgaagat gaggatgata ggaaacagat 60 tgatgagcta ctaggcaaag ttgtatgtgt agattacatt agtttggata aaaagaaagc 120 actgtggttt cctgcattg 139 13 80 DNA Homo sapiens 13 gtggtttgtc ctgattgtag tgatgagatt gctgtaaaaa aggacaatat tcttgttcga 60 tctttcaaag atggaaaatt 80 14 77 DNA Homo sapiens 14 tacttcagtt ccaagaaaag atgtccatga aattactagt gacactgcac caaagcctga 60 tgctgtttta aagcaag 77 15 155 DNA Homo sapiens 15 cctttgaaca ggcacttgaa tttcacaaaa gtagaactat tcctgctaac tggaagactg 60 aattgaaaga agatagctct agcagtgaag cagaggaaga agaggaggag gaagatgatg 120 aaaaagaaaa ggaggataat agcagtgaag aagag 155 16 73 DNA Homo sapiens 16 gaagaaatag aaccatttcc agaagaaagg gagaactttc ttcagcaatt gtacaaattt 60 atggaagata gag 73 17 95 DNA Homo sapiens 17 gtacacctat taacaaacga cctgtacttg gatatcgaaa tttgaatctc tttaagttat 60 tcagacttgt acacaaactt ggaggatttg ataat 95 18 98 DNA Homo sapiens 18 attgaaagtg gagctgtttg gaaacaagtc taccaagatc ttggaatccc tgtcttaaat 60 tcagctgcag gatacaatgt taaatgtgct tataaaaa 98 19 244 DNA Homo sapiens 19 atacttatat ggttttgagg agtactgtag atcagccaac attgaatttc agatggcatt 60 gccagagaaa gttgttaaca agcaatgtaa ggagtgtgaa aatgtaaaag aaataaaagt 120 taaggaggaa aatgaaacag agatcaaaga aataaagatg gaggaggaga ggaatataat 180 accaagagaa gaaaagccta ttgaggatga aattgaaaga aaagaaaata ttaagccctc 240 tctg 244 20 176 DNA Homo sapiens 20 ggaagtaaaa agaatttatt agaatctata cctacacatt ctgatcagga aaaagaagtt 60 aacattaaaa aaccagaaga caatgaaaat ctggacgaca aagatgatga cacaactagg 120 gtagatgaat ccctcaacat aaaggtagaa gctgaggaag aaaaagcaaa atctgg 176 21 258 DNA Homo sapiens 21 agatgaaacg aataaagaag aagatgaaga tgatgaagaa gcagaagagg aggaggagga 60 ggaagaagaa gaagaggatg aagatgatga tgacaacaat gaggaagagg agtttgagtg 120 ctatccacca ggcatgaaag tccaagtgcg gtatggacga gggaaaaatc aaaaaatgta 180 tgaagctagt attaaagatt ctgatgttga aggtggagag gtcctttact tggtgcatta 240 ctgcggatgg aatgtgag 258 22 85 DNA Homo sapiens 22 atacgatgaa tggattaaag cagataaaat agtaagacct gctgataaaa atgtgccaaa 60 gataaaacat cggaagaaaa taaag 85 23 199 DNA Homo sapiens 23 aataaattag acaaagaaaa agacaaagat gaaaaatact ctccaaaaaa ctgtaaactt 60 cggcgcttgt ccaaaccacc atttcagaca aatccatctc ctgaaatggt atccaaactg 120 gatctcactg atgccaaaaa ctctgatact gctcatatta agtccataga aattacttcg 180 atccttaatg gacttcaag 199 24 1209 DNA Homo sapiens 24 cttctgaaag ttctgctgaa gacagtgagc aggaagatga gagaggtgct caagacatgg 60 ataataatgg caaagaggaa tctaagattg atcatttgac caacaacaga aatgatctta 120 tttcaaagga ggaacagaac agttcatctt tgctagaaga aaacaaagtt catgcagatt 180 tggtaatatc caaaccagtg tcaaaatctc cagaaagatt aaggaaagat atagaagtat 240 tatccgaaga tactgattat gaagaagatg aagtcacaaa aaagagaaag gatgtcaaga 300 aggacacaac agataaatct tcaaaaccac aaataaaacg tggtaaaaga aggtattgca 360 atacagaaga gtgtctaaaa actggatcac ctggcaaaaa ggaagagaag gccaagaaca 420 aagaatcact ttgcatggaa aacagtagca acagctcttc agatgaagat gaagaagaaa 480 caaaagcaaa gatgacacca actaagaaat acaatggttt ggaggaaaaa agaaaatctc 540 tacggacaac tggtttctat tcaggatttt cagaagtggc agaaaaaagg attaaacttt 600 taaataactc tgatgaaaga cttcaaaaca gcagggccaa agatcgaaaa gatgtctggt 660 caagtattca gggacagtgg cctaaaaaaa cgctgaaaga gcttttttca gactctgata 720 ctgaggctgc agcttcccca ccgcatcctg ccccagagga gggggtggca gaggagtcac 780 tgcagactgt ggctgaagag gagagttgtt cacccagtgt agaactagaa aaaccacctc 840 cagtcaatgt cgatagtaaa cccattgaag aaaaaacagt agaggtcaat gacagaaaag 900 cagaatttcc aagtagtggc agtaattcag tgctaaatac ccctcctact acacctgaat 960 cgccttcatc agtcactgta acagaaggca gccggcagca gtcttctgta acagtatcag 1020 aaccactggc tccaaaccaa gaagaggttc gaagtatcaa gagtgaaact gatagcacaa 1080 ttgaggtgga tagtgttgct ggggagctcc aagacctcca gtctgaaggg aatagctcgc 1140 cagcaggttt tgatgccagt gtgagctcaa gcagtagtaa tcagccagaa ccagaacatc 1200 ctgaaaaag 1209 25 114 DNA Homo sapiens 25 cctgtacagg tcagaaaaga gtgaaagatg ctcagggagg aggaagttca tcaaaaaagc 60 agaaaagaag ccataaagca acagtggtaa acaacaaaaa gaagggaaaa ggca 114 26 216 DNA Homo sapiens 26 caaatagtag tgatagtgaa gaactttcag ctggtgaaag tataactaag agtcagccag 60 tcaaatcagt ttccactgga atgaagtctc atagtaccaa atctcccgca aggacgcagt 120 ctccaggaaa atgtggaaag aatggtgata aggatcctga tctcaaggaa cccagtaatc 180 gattacccaa agtttacaaa tggagttttc agatgt 216 27 147 DNA Homo sapiens 27 cggacctgga aaatatgaca agtgccgaac gcatcacaat tcttcaagaa aaacttcaag 60 aaatcagaaa acattatctg tcattaaaat ctgaagtagc ttccattgat cggaggagaa 120 agcgtttaaa gaagaaagag agagaaa 147 28 1750 DNA Homo sapiens 28 gtgctgctac atcctcatcc tcctcttcac cttcatccag ttccataaca gctgctgtta 60 tgttaacttt agctgaaccg tcaatgtcca gcgcatcaca aaatggaatg tcagttgagt 120 gcaggtgaca gcaggacttg ctaaagcact ttgcacttaa tggctgttga gggccacttt 180 ttttttatac tgcacagtgg cacaaaaaaa tatcagacaa gcactatttt atatttaaaa 240 attgtttctt gacaagctga cttggcactt aagtgcactt ttttatgaag aaaaagtaca 300 atgaactgct tttcctcaag caataattgt ttccaacttg tctgggaatt gtgtgtctgg 360 taactggaag gccttccact gtggcaaatg gaggcttttc actgcctgta gagacaatac 420 agtaagcata gttaaggggt gggtcagaac atgttaagat aacttactgt atatgtattc 480 ccttgtattt tgttaaagct ggaacatttg atatttttcc atttatttat gaaaaaatat 540 gaacctattt tcatttgtac aaggtaattg ttttttaaag caagtcacct tagggtggct 600 ttaattgtat aagtcaagca catgtaataa attcaaaacc tgcagttaac aggatattag 660 acatcaatcc tggtaaccaa atattaaaga ttctctttaa aaaagactga acatgtttac 720 aggtttgaat taggctaaaa ggtcttgcag tggcttttca tggcccttca aattggaatg 780 gaactactgt actttgccat ttttctataa atcagtattt ttttttaatt ttgatataca 840 ttgtgtgaaa aaagaaaatg gctaataaac tgtattaaat cttaaacaat gtataaagat 900 tgtacttagc cagttcaaag tgtatattta ttcataatga attataacag ttatattttt 960 gtgttttctt gtaaatgttt cttttccctt aaatacagat aattcatttg tattgcttat 1020 tttattatga gctacaacaa aaggacttca ggaacaagta atgtattagt atggttcaag 1080 attgttgata ggaactgtct caaaaggatg gtggttattt taaatataaa tagctaatgg 1140 gggtggtagg cctataaaat taaatgcctt gtataaaatc caaaatgaat gcaaaattgt 1200 tttcacttgt attgacttta tgttgtatga ttccaatctc tgttctgttt ggcacttgta 1260 tttaattctt cacctttgta agacatttgt atattgtgga tgtgttcatt caagctattt 1320 aatatctggc actgttaata cacagtactt tattgtacag actgttttac tgttttaatt 1380 gtagttctgt gtactttttt tggatggggc tggcatgttt tctttgtttc ctggcaatac 1440 gacgtgggaa tttcaatgcg ttttgttgta gatgctaacg tgtcagaatc ctttacattc 1500 aacttttcta agaaaagcat tttcagtctt gtagtgtgtg cttacagtaa ctaattttgt 1560 tgaaaatggt ttcaagttat tcaaatttgt acaggactgt aaagatttgt tgacagcaaa 1620 atgttgaaga aaaaagctta tagaataaaa gctataaagt atatattagg atctgcaaac 1680 aatgaagaat tatgtaatat attgtacaaa tgtaagcaaa ggctctgaaa taaaatgcca 1740 tagtttgtga 1750 29 1312 PRT Homo sapiens CARBOHYD 294..296 potential 29 Met Lys Ala Leu Asp Glu Pro Pro Tyr Leu Thr Val Gly Thr Asp Val 1 5 10 15 Ser Ala Lys Tyr Arg Gly Ala Phe Cys Glu Ala Lys Ile Lys Thr Ala 20 25 30 Lys Arg Leu Val Lys Val Lys Val Thr Phe Arg His Asp Ser Ser Thr 35 40 45 Val Glu Val Gln Asp Asp His Ile Lys Gly Pro Leu Lys Val Gly Ala 50 55 60 Ile Val Glu Val Lys Asn Leu Asp Gly Ala Tyr Gln Glu Ala Val Ile 65 70 75 80 Asn Lys Leu Thr Asp Ala Ser Trp Tyr Thr Val Val Phe Asp Asp Gly 85 90 95 Asp Glu Lys Thr Leu Arg Arg Ser Ser Leu Cys Leu Lys Gly Glu Arg 100 105 110 His Phe Ala Glu Ser Glu Thr Leu Asp Gln Leu Pro Leu Thr Asn Pro 115 120 125 Glu His Phe Gly Thr Pro Val Ile Gly Lys Lys Thr Asn Arg Gly Arg 130 135 140 Arg Ser Asn His Ile Pro Glu Glu Glu Ser Ser Ser Ser Ser Ser Asp 145 150 155 160 Glu Asp Glu Asp Asp Arg Lys Gln Ile Asp Glu Leu Leu Gly Lys Val 165 170 175 Val Cys Val Asp Tyr Ile Ser Leu Asp Lys Lys Lys Ala Leu Trp Phe 180 185 190 Pro Ala Leu Val Val Cys Pro Asp Cys Ser Asp Glu Ile Ala Val Lys 195 200 205 Lys Asp Asn Ile Leu Val Arg Ser Phe Lys Asp Gly Lys Phe Thr Ser 210 215 220 Val Pro Arg Lys Asp Val His Glu Ile Thr Ser Asp Thr Ala Pro Lys 225 230 235 240 Pro Asp Ala Val Leu Lys Gln Ala Phe Glu Gln Ala Leu Glu Phe His 245 250 255 Lys Ser Arg Thr Ile Pro Ala Asn Trp Lys Thr Glu Leu Lys Glu Asp 260 265 270 Ser Ser Ser Ser Glu Ala Glu Glu Glu Glu Glu Glu Glu Asp Asp Glu 275 280 285 Lys Glu Lys Glu Xaa Asn Ser Ser Glu Glu Glu Glu Glu Ile Glu Pro 290 295 300 Phe Pro Glu Glu Arg Glu Asn Phe Leu Gln Gln Leu Tyr Lys Phe Met 305 310 315 320 Glu Asp Arg Gly Thr Pro Ile Asn Lys Arg Pro Val Leu Gly Tyr Arg 325 330 335 Asn Leu Asn Leu Phe Lys Leu Phe Arg Leu Val His Lys Leu Gly Gly 340 345 350 Phe Asp Asn Ile Glu Ser Gly Ala Val Trp Lys Gln Val Tyr Gln Asp 355 360 365 Leu Gly Ile Pro Val Leu Asn Ser Ala Ala Gly Tyr Asn Val Lys Cys 370 375 380 Ala Tyr Lys Lys Tyr Leu Tyr Gly Phe Glu Glu Tyr Cys Arg Ser Ala 385 390 395 400 Asn Ile Glu Phe Gln Met Ala Leu Pro Glu Lys Val Val Asn Lys Gln 405 410 415 Cys Lys Glu Cys Glu Asn Val Lys Glu Ile Lys Val Lys Glu Glu Asn 420 425 430 Glu Thr Glu Ile Lys Glu Ile Lys Met Glu Glu Glu Arg Asn Ile Ile 435 440 445 Pro Arg Glu Glu Lys Pro Ile Glu Asp Glu Ile Glu Arg Lys Glu Asn 450 455 460 Ile Lys Pro Ser Leu Gly Ser Lys Lys Asn Leu Leu Glu Ser Ile Pro 465 470 475 480 Thr His Ser Asp Gln Glu Lys Glu Val Asn Ile Lys Lys Pro Glu Asp 485 490 495 Asn Glu Asn Leu Asp Asp Lys Asp Asp Asp Thr Thr Arg Val Asp Glu 500 505 510 Ser Leu Asn Ile Lys Val Glu Ala Glu Glu Glu Lys Ala Lys Ser Gly 515 520 525 Asp Glu Thr Asn Lys Glu Glu Asp Glu Asp Asp Glu Glu Ala Glu Glu 530 535 540 Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Glu Asp Asp Asp Asp Asn 545 550 555 560 Asn Glu Glu Glu Glu Phe Glu Cys Tyr Pro Pro Gly Met Lys Val Gln 565 570 575 Val Arg Tyr Gly Arg Gly Lys Asn Gln Lys Met Tyr Glu Ala Ser Ile 580 585 590 Lys Asp Ser Asp Val Glu Gly Gly Glu Val Leu Tyr Leu Val His Tyr 595 600 605 Cys Gly Trp Asn Val Arg Tyr Asp Glu Trp Ile Lys Ala Asp Lys Ile 610 615 620 Val Arg Pro Ala Asp Lys Asn Val Pro Lys Ile Lys His Arg Lys Lys 625 630 635 640 Ile Lys Asn Lys Leu Asp Lys Glu Lys Asp Lys Asp Glu Lys Tyr Ser 645 650 655 Pro Lys Asn Cys Lys Leu Arg Arg Leu Ser Lys Pro Pro Phe Gln Thr 660 665 670 Asn Pro Ser Pro Glu Met Val Ser Lys Leu Asp Leu Thr Asp Ala Lys 675 680 685 Asn Ser Asp Thr Ala His Ile Lys Ser Ile Glu Ile Thr Ser Ile Leu 690 695 700 Asn Gly Leu Gln Ala Ser Glu Ser Ser Ala Glu Asp Ser Glu Gln Glu 705 710 715 720 Asp Glu Arg Gly Ala Gln Asp Met Asp Asn Asn Gly Lys Glu Glu Ser 725 730 735 Lys Ile Asp His Leu Thr Asn Asn Arg Asn Asp Leu Ile Ser Lys Glu 740 745 750 Glu Gln Asn Ser Ser Ser Leu Leu Glu Glu Asn Lys Val His Ala Asp 755 760 765 Leu Val Ile Ser Lys Pro Val Ser Lys Ser Pro Glu Arg Leu Arg Lys 770 775 780 Asp Ile Glu Val Leu Ser Glu Asp Thr Asp Tyr Glu Glu Asp Glu Val 785 790 795 800 Thr Lys Lys Arg Lys Asp Val Lys Lys Asp Thr Thr Asp Lys Ser Ser 805 810 815 Lys Pro Gln Ile Lys Arg Gly Lys Arg Arg Tyr Cys Asn Thr Glu Glu 820 825 830 Cys Leu Lys Thr Gly Ser Pro Gly Lys Lys Glu Glu Lys Ala Lys Asn 835 840 845 Lys Glu Ser Leu Cys Met Glu Asn Ser Ser Asn Ser Ser Ser Asp Glu 850 855 860 Asp Glu Glu Glu Thr Lys Ala Lys Met Thr Pro Thr Lys Lys Tyr Asn 865 870 875 880 Gly Leu Glu Glu Lys Arg Lys Ser Leu Arg Thr Thr Gly Phe Tyr Ser 885 890 895 Gly Phe Ser Glu Val Ala Glu Lys Arg Ile Lys Leu Leu Asn Asn Ser 900 905 910 Asp Glu Arg Leu Gln Asn Ser Arg Ala Lys Asp Arg Lys Asp Val Trp 915 920 925 Ser Ser Ile Gln Gly Gln Trp Pro Lys Lys Thr Leu Lys Glu Leu Phe 930 935 940 Ser Asp Ser Asp Thr Glu Ala Ala Ala Ser Pro Pro His Pro Ala Pro 945 950 955 960 Glu Glu Xaa Val Ala Glu Glu Ser Xaa Gln Thr Val Ala Glu Glu Glu 965 970 975 Ser Cys Ser Pro Ser Val Glu Leu Glu Lys Pro Pro Pro Val Asn Val 980 985 990 Asp Ser Lys Pro Ile Glu Glu Lys Thr Val Glu Val Asn Asp Arg Lys 995 1000 1005 Ala Glu Phe Pro Ser Ser Gly Ser Asn Ser Val Leu Asn Thr Pro Pro 1010 1015 1020 Thr Thr Pro Glu Ser Pro Ser Ser Val Thr Val Thr Glu Gly Ser Arg 1025 1030 1035 1040 Gln Gln Ser Ser Val Thr Val Ser Glu Pro Leu Ala Pro Asn Gln Glu 1045 1050 1055 Glu Val Arg Ser Ile Lys Ser Glu Thr Asp Ser Thr Ile Glu Val Asp 1060 1065 1070 Ser Val Ala Gly Glu Leu Gln Asp Leu Gln Ser Glu Gly Asn Ser Ser 1075 1080 1085 Pro Ala Gly Phe Asp Ala Ser Val Ser Ser Ser Ser Ser Asn Gln Pro 1090 1095 1100 Glu Pro Glu His Pro Glu Lys Ala Cys Thr Gly Gln Lys Arg Val Lys 1105 1110 1115 1120 Asp Ala Gln Gly Gly Gly Ser Ser Ser Lys Lys Gln Lys Arg Ser His 1125 1130 1135 Lys Ala Thr Val Val Asn Asn Lys Lys Lys Gly Lys Gly Thr Asn Ser 1140 1145 1150 Ser Asp Ser Glu Glu Leu Ser Ala Gly Glu Ser Ile Thr Lys Ser Gln 1155 1160 1165 Pro Val Lys Ser Val Ser Thr Gly Met Lys Ser His Ser Thr Lys Ser 1170 1175 1180 Pro Ala Arg Thr Gln Ser Pro Gly Lys Cys Gly Lys Asn Gly Asp Lys 1185 1190 1195 1200 Asp Pro Asp Leu Lys Glu Pro Ser Asn Arg Leu Pro Lys Val Tyr Lys 1205 1210 1215 Trp Ser Phe Gln Met Ser Asp Leu Glu Asn Met Thr Ser Ala Glu Arg 1220 1225 1230 Ile Thr Ile Leu Gln Glu Lys Leu Gln Glu Ile Arg Lys His Tyr Leu 1235 1240 1245 Ser Leu Lys Ser Glu Val Ala Ser Ile Asp Arg Arg Arg Lys Arg Leu 1250 1255 1260 Lys Lys Lys Glu Arg Glu Ser Ala Ala Thr Ser Ser Ser Ser Ser Ser 1265 1270 1275 1280 Pro Ser Ser Ser Ser Ile Thr Ala Ala Val Met Leu Thr Leu Ala Glu 1285 1290 1295 Pro Ser Met Ser Ser Ala Ser Gln Asn Gly Met Ser Val Glu Cys Arg 1300 1305 1310 30 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-124-273 30 attcacttct taatacccta gatattatta ctgttactgg wttttat 47 31 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-127-261 31 ttcagtatac aagagtttaa tttaaaactt tataagttta tgaagaa 47 32 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-128-60 32 aaaattgctt gtgtgtgctc ccacgtgtgt gtgtgtgcct gtttacc 47 33 48 DNA Homo Sapiens allele 1..48 polymorphic fragment 5-129-144 33 cttctcttat aattaaaaaa aatatatagt acttcagttc caagaaaa 48 34 39 DNA Homo Sapiens allele 1..39 polymorphic fragment 5-130-257 34 agatgatgaa aaagaaaagg aggataatag cagtgaaga 39 35 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-130-276 35 gaggataata gcagtgaaga agaagtaagt gaaaacagtt gatacct 47 36 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-131-395 36 cctagcatag cgcctgtcac gtaacaagta gaaykgagga atttgat 47 37 50 DNA Homo Sapiens allele 1..50 polymorphic fragment 5-133-375 37 ttttctaaag tgtattctat gaatactaga tctatgagaa attctgtgaa 50 38 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-135-155 38 tattttccat atcctctata aagttccaaa atcaatatat tgtataa 47 39 50 DNA Homo Sapiens allele 1..50 polymorphic fragment 5-135-198 39 ataatattat tctttattat ttgttttttt cttcattaag tgctactttt 50 40 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-135-357 40 ggttgatacc tcctgttgct aagagataaa ccatggatat aggttga 47 41 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-136-174 41 ccagaagaca atgaaaatct ggacgacaaa gatgatgaca caactag 47 42 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-140-120 42 ccttatgata aattacgaca tacctttttt cttaacctag aataaat 47 43 49 DNA Homo Sapiens allele 1..49 polymorphic fragment 5-140-348 43 ggacttcaag gtaaacataa caatcgttct gttgcatgca agtatttga 49 44 48 DNA Homo Sapiens allele 1..48 polymorphic fragment 5-140-361 44 aaacataaca atcgttctgt tgcatgcaag tatttgattt taatttat 48 45 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-143-101 45 aggagggggt ggcagaggag tcaatgcaga ctgtggctga agaggag 47 46 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-143-84 46 accgcatcct gccccagagg aggaggtggc agaggagtca ctgcaga 47 47 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-145-24 47 tagtaagttc tgtgacaact tataaatgtc ataaagaaca tgtagtt 47 48 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-148-352 48 tgcttttcct caagcaataa ttggttccaa cttgtctggg aattgtg 47 49 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 99-1437-325 49 caagagctga catttactgc atacttaatt tgtgccgaac actgaac 47 50 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 99-1442-224 50 attaatctca gtcatatttt ggggtttttt tcttctctta taattaa 47 51 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-124-273, variant version of SEQ ID30 51 attcacttct taatacccta gatgttatta ctgttactgg wttttat 47 52 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-127-261, variant version of SEQ ID31 52 ttcagtatac aagagtttaa tttcaaactt tataagttta tgaagaa 47 53 45 DNA Homo Sapiens allele 1..45 polymorphic fragment 5-128-60, variant version of SEQ ID32 53 aaaattgctt gtgtgtgctc ccacgtgtgt gtgtgcctgt ttacc 45 54 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-129-144, variant version of SEQ ID33 54 cttctcttat aattaaaaaa aaatatagta cttcagttcc aagaaaa 47 55 39 DNA Homo Sapiens allele 1..39 polymorphic fragment 5-130-257, variant version of SEQ ID34 55 agatgatgaa aaagaaaagg agggtaatag cagtgaaga 39 56 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-130-276, variant version of SEQ ID35 56 gaggataata gcagtgaaga agaggtaagt gaaaacagtt gatacct 47 57 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-131-395, variant version of SEQ ID36 57 cctagcatag cgcctgtcac gtatcaagta gaaykgagga atttgat 47 58 49 DNA Homo Sapiens allele 1..49 polymorphic fragment 5-133-375, variant version of SEQ ID37 58 ttttctaaag tgtattctat gatactagat ctatgagaaa ttctgtgaa 49 59 48 DNA Homo Sapiens allele 1..48 polymorphic fragment 5-135-155, variant version of SEQ ID38 59 tattttccat atcctctata aaagttccaa aatcaatata ttgtataa 48 60 46 DNA Homo Sapiens allele 1..46 polymorphic fragment 5-135-198, variant version of SEQ ID39 60 ataatattat tctttattat ttttttcttc attaagtgct actttt 46 61 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-135-357, variant version of SEQ ID40 61 ggttgatacc tcctgttgct aagggataaa ccatggatat aggttga 47 62 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-136-174, variant version of SEQ ID41 62 ccagaagaca atgaaaatct ggatgacaaa gatgatgaca caactag 47 63 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-140-120, variant version of SEQ ID42 63 ccttatgata aattacgaca tacttttttt cttaacctag aataaat 47 64 48 DNA Homo Sapiens allele 1..48 polymorphic fragment 5-140-348, variant version of SEQ ID43 64 ggacttcaag gtaaacataa catcgttctg ttgcatgcaa gtatttga 48 65 46 DNA Homo Sapiens allele 1..46 polymorphic fragment 5-140-361, variant version of SEQ ID44 65 aaacataaca atcgttctgt tgtgcaagta tttgatttta atttat 46 66 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-143-101, variant version of SEQ ID45 66 aggagggggt ggcagaggag tcactgcaga ctgtggctga agaggag 47 67 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-143-84, variant version of SEQ ID46 67 accgcatcct gccccagagg agggggtggc agaggagtca ctgcaga 47 68 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-145-24, variant version of SEQ ID47 68 tagtaagttc tgtgacaact tatgaatgtc ataaagaaca tgtagtt 47 69 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 5-148-352, variant version of SEQ ID48 69 tgcttttcct caagcaataa ttgtttccaa cttgtctggg aattgtg 47 70 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 99-1437-325, variant version of SEQ ID49 70 caagagctga catttactgc atatttaatt tgtgccgaac actgaac 47 71 47 DNA Homo Sapiens allele 1..47 polymorphic fragment 99-1442-224, variant version of SEQ ID50 71 attaatctca gtcatatttt gggttttttt tcttctctta taattaa 47 72 19 DNA Artificial Sequence primer_bind 1..19 upstream amplification primer for SEQ 30, SEQ 51 72 aaaagaaaac aaacccagg 19 73 19 DNA Artificial Sequence primer_bind 1..19 upstream amplification primer for SEQ 31, SEQ 52 73 ataagagttt gggaatacc 19 74 18 DNA Artificial Sequence primer_bind 1..18 upstream amplification primer for SEQ 32, SEQ 53 74 tggaaggatg taggatgc 18 75 18 DNA Artificial Sequence primer_bind 1..18 upstream amplification primer for SEQ 33, SEQ 54 75 gctactctgt gtgcaatc 18 76 20 DNA Artificial Sequence primer_bind 1..20 upstream amplification primer for SEQ 34, SEQ 55, SEQ 35, SEQ 56 76 caaacaataa atgtcagtgg 20 77 19 DNA Artificial Sequence primer_bind 1..19 upstream amplification primer for SEQ 36, SEQ 57 77 ggttttgaac agcttagtg 19 78 18 DNA Artificial Sequence primer_bind 1..18 upstream amplification primer for SEQ 37, SEQ 58 78 tcttttgagt ctaggacc 18 79 18 DNA Artificial Sequence primer_bind 1..18 upstream amplification primer for SEQ 38, SEQ 59, SEQ 39, SEQ 60, SEQ 40, SEQ 61 79 aaagatggag gaggagag 18 80 19 DNA Artificial Sequence primer_bind 1..19 upstream amplification primer for SEQ 41, SEQ 62 80 tgttgctaag agataaacc 19 81 19 DNA Artificial Sequence primer_bind 1..19 upstream amplification primer for SEQ 42, SEQ 63, SEQ 43, SEQ 64, SEQ 44, SEQ 65 81 gggcttctta tgttctttc 19 82 20 DNA Artificial Sequence primer_bind 1..20 upstream amplification primer for SEQ 45, SEQ 66, SEQ 46, SEQ 67 82 gcctaaaaaa acgctgaaag 20 83 19 DNA Artificial Sequence primer_bind 1..19 upstream amplification primer for SEQ 47, SEQ 68 83 tagtaagttc tgtgacaac 19 84 18 DNA Artificial Sequence primer_bind 1..18 upstream amplification primer for SEQ 48, SEQ 69 84 gtttggtctc ccttttcc 18 85 20 DNA Artificial Sequence primer_bind 1..20 upstream amplification primer for SEQ 49, SEQ 70 85 caaaagaaac tcacaaagac 20 86 18 DNA Artificial Sequence primer_bind 1..18 upstream amplification primer for SEQ 50, SEQ 71 86 tctggtattt gggaacac 18 87 19 DNA Artificial Sequence primer_bind 1..19 downstream amplification primer for SEQ 30, SEQ 51 87 gtggaaagat aaaatccag 19 88 19 DNA Artificial Sequence primer_bind 1..19 downstream amplification primer for SEQ 31, SEQ 52 88 caagattttg cctttcctg 19 89 19 DNA Artificial Sequence primer_bind 1..19 downstream amplification primer for SEQ 32, SEQ 53 89 gaaaaacagt gactctttg 19 90 19 DNA Artificial Sequence primer_bind 1..19 downstream amplification primer for SEQ 33, SEQ 54 90 aataaaggtc acaaggaac 19 91 20 DNA Artificial Sequence primer_bind 1..20 downstream amplification primer for SEQ 34, SEQ 55, SEQ 35, SEQ 56 91 atgaggatct caaatacaag 20 92 19 DNA Artificial Sequence primer_bind 1..19 downstream amplification primer for SEQ 36, SEQ 57 92 gctatcaaat tcctcattc 19 93 20 DNA Artificial Sequence primer_bind 1..20 downstream amplification primer for SEQ 37, SEQ 58 93 cacaaaaact ttcttcacag 20 94 19 DNA Artificial Sequence primer_bind 1..19 downstream amplification primer for SEQ 38, SEQ 59, SEQ 39, SEQ 60, SEQ 40, SEQ 61 94 ttccctagaa aaagatcag 19 95 19 DNA Artificial Sequence primer_bind 1..19 downstream amplification primer for SEQ 41, SEQ 62 95 caaatttcta tcagtaggg 19 96 20 DNA Artificial Sequence primer_bind 1..20 downstream amplification primer for SEQ 42, SEQ 63, SEQ 43, SEQ 64, SEQ 44, SEQ 65 96 acagtagttg gtaatgtcac 20 97 18 DNA Artificial Sequence primer_bind 1..18 downstream amplification primer for SEQ 45, SEQ 66, SEQ 46, SEQ 67 97 agcaacacta tccacctc 18 98 20 DNA Artificial Sequence primer_bind 1..20 downstream amplification primer for SEQ 47, SEQ 68 98 gttttttaaa gcaagtagcc 20 99 20 DNA Artificial Sequence primer_bind 1..20 downstream amplification primer for SEQ 48, SEQ 69 99 aaagcctcca tttgccacag 20 100 21 DNA Artificial Sequence primer_bind 1..21 downstream amplification primer for SEQ 49, SEQ 70 100 attctcattc tctcattttc c 21 101 19 DNA Artificial Sequence primer_bind 1..19 downstream amplification primer for SEQ 50, SEQ 71 101 aataaaggtc acaaggaac 19 102 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-124-273.mis1 102 acttcttaat accctagat 19 103 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-127-261.mis1 103 gtatacaaga gtttaattt 19 104 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-128-60. mis1 104 tgcttgtgtg tgctcccac 19 105 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-129-144. mis1 105 ctcttataat taaaaaaaa 19 106 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-130-257.mis1 106 gatgaaaaag aaaaggagg 19 107 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-130-276. mis1 107 ataatagcag tgaagaaga 19 108 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-131-395.mis1 108 gcatagcgcc tgtcacgta 19 109 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-133-375.mis1 109 tctaaagtgt attctatga 19 110 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-135-155.mis1 110 tttccatatc ctctataaa 19 111 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-135-198. mis1 111 atattattct ttattattt 19 112 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-135-357. mis1 112 gatacctcct gttgctaag 19 113 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-136-174.mis1 113 aagacaatga aaatctgga 19 114 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-140-120.mis1 114 atgataaatt acgacatac 19 115 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-140-348.mis1 115 cttcaaggta aacataaca 19 116 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-140-361. mis1 116 cataacaatc gttctgttg 19 117 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-143-101. mis1 117 gggggtggca gaggagtca 19 118 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-143-84. mis1 118 catcctgccc cagaggagg 19 119 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-145-24.mis1 119 aagttctgtg acaacttat 19 120 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-148-352. mis1 120 tttcctcaag caataattg 19 121 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 99-1437-325.mis1 121 agctgacatt tactgcata 19 122 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 99-1442-224.mis1 122 atctcagtca tattttggg 19 123 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-124-273.mis2 123 aawccagtaa cagtaataa 19 124 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-127-261.mis2 124 tcataaactt ataaagttt 19 125 15 DNA Artificial Sequence primer_bind 1..15 potential microsequencing oligo for 5-130-257.mis2 125 tcttcactgc tatta 15 126 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-130-276.mis2 126 atcaactgtt ttcacttac 19 127 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-131-395.mis2 127 aattcctcmr ttctacttg 19 128 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-135-357.mis2 128 cctatatcca tggtttatc 19 129 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-136-174.mis2 129 ttgtgtcatc atctttgtc 19 130 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-140-120.mis2 130 attctaggtt aagaaaaaa 19 131 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-143-101.mis2 131 tcttcagcca cagtctgca 19 132 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 5-143-84.mis2 132 cagtgactcc tctgccacc 19 133 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-145-24.mis2 133 acatgttctt tatgacatt 19 134 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 5-148-352.mis2 134 attcccagac aagttggaa 19 135 19 DNA Artificial Sequence primer_bind 1..19 potential microsequencing oligo for 99-1437-325.mis2 135 agtgttcggc acaaattaa 19 136 19 DNA Artificial Sequence primer_bind 1..19 microsequencing oligo for 99-1442-224.mis2 136 ttataagaga agaaaaaaa 19 137 27 DNA Artificial Sequence misc_binding 1..27 amplification oligonucleotide hRBBP1.5 137 cccttgatga gcctccctat ttgacag 27 138 30 DNA Artificial Sequence misc_binding 1..30 amplification oligonucleotide hRBBP1.3 138 cgcattgaaa ttcccacgtc gtattgccag 30 139 18 DNA Artificial Sequence misc_binding 1..18 sequencing oligonucleotide PrimerPU 139 tgtaaaacga cggccagt 18 140 18 DNA Artificial Sequence misc_binding 1..18 sequencing oligonucleotide PrimerRP 140 caggaaacag ctatgacc 18 

What is claimed is:
 1. A purified or isolated polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 1 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 1: 1481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842-146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, and 161453-162450.
 2. A purified or isolated polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 4 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579.
 3. A purified or isolated nucleic acid comprising a nucleotide sequence selected from the group of SEQ ID Nos. 5-28, a sequence complementary thereto or a fragment or a variant thereof.
 4. A purified or isolated nucleic acid comprising a combination of at least two polynucleotides selected from the group consisting of SEQ ID Nos. 5-28, wherein the polynucleotides are arranged within the nucleic acid, from the 5′ end to the 3′ end of said nucleic acid, in the same order than in SEQ ID No.
 1. 5. An oligonucleotide of at least 8 nucleotides in length that hybridizes under stringent hybridization conditions with a nucleic acid selected from the group consisting of the nucleotide sequences 1481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842 146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, and 161453-162450 of SEQ ID No. 1 or a sequence complementary thereto.
 6. An oligonucleotide of at least 8 nucleotides in length that hybridizes under stringent hybridization conditions with a nucleic acid selected from the group consisting of the nucleotide sequences 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579 of SEQ ID No. 4 or a sequence complementary thereto.
 7. An isolated, purified, or recombinant polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos. 1 and 4 and the complement thereof, wherein said span includes a biallelic marker of RBP-7.
 8. A polynucleotide according to claim 7, wherein said biallelic marker of RBP-7 is selected from the group consisting of A1 to A21, and the complements thereof.
 9. A polynucleotide according to claim 7, wherein said contiguous span is 18 to 47 nucleotides in length and said biallelic marker is within 4 nucleotides of the center of said polynucleotide.
 10. A polynucleotide according to claim 9, wherein said polynucleotide consists of said contiguous span and said contiguous span is 25 nucleotides in length and said biallelic marker is at the center of said polynucleotide.
 11. A polynucleotide according to claim 9, wherein said polynucleotide consists essentially of a sequence selected from the sequences SEQ ID Nos. 30-71 and the complementary sequences thereto.
 12. A polynucleotide according to claim 7, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide and said biallelic marker is present at the 3′ end of said polynucleotide.
 13. An isolated, purified, or recombinant polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos. 1 and 4 and the complement thereof, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide, and wherein the 3′ end of said polynucleotide is located within 20 nucleotides upstream of a biallelic marker of RBP-7 in said sequence.
 14. A polynucleotide according to claim 13, wherein the 3′ end of said polynucleotide is located 1 nucleotide upstream of said biallelic marker of RBP-7 in said sequence.
 15. A polynucleotide according to claim 14, wherein said polynucleotide consists essentially of a sequence selected from the sequences of SEQ ID Nos. 102-136.
 16. An isolated, purified, or recombinant polynucleotide consisting essentially of a sequence selected from the sequences of SEQ ID Nos. 72-101.
 17. An isolated, purified, or recombinant polynucleotide which encodes a polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ID No.
 29. 18. An isolated, purified, or recombinant polynucleotide for use in hybridization assays, sequencing assays, and enzyme-based mismatch detection assays for determining the identity of the nucleotide at a biallelic marker of RBP-7.
 19. A solid support having a polynucleotide attached thereto, wherein said polynucleotide is selected from the group consisting of: a) a polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 1 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 1: 1481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842-146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, and 161453-162450; b) a polynucleotides comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 4 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579; c) a polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos. 1 and 4 and the complement thereof, wherein said span includes a biallelic marker of RBP-7; d) a polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos. 1 and 4 and the complement thereof, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide, and wherein the 3′ end of said polynucleotide is located within 20 nucleotides upstream of a biallelic marker of RBP-7 in said sequence; e) a polynucleotide consisting essentially of a sequence selected from the sequences of SEQ ID Nos. 72-101; f) a polynucleotide which encodes a polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ID No. 29; and g) a polynucleotide for use in hybridization assays, sequencing assays, and enzyme-based mismatch detection assays for determining the identity of the nucleotide at a biallelic marker of RBP-7.
 20. An array of polynucleotides comprising at least one polynucleotide according to claim
 19. 21. An array according to claim 20, wherein said array is addressable.
 22. A polynucleotide having a label thereon, wherein said polynucleotide is selected from the group consisting of: a) a polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 1 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 1: 1481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842-146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, and 161453-162450; b) a polynucleotides comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 4 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579; c) a polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos. 1 and 4 and the complement thereof, wherein said span includes a biallelic marker of RBP-7; d) a polynucleotide consisting essentially of a contiguous span of 8 to 50 nucleotides of any one of SEQ ID Nos. 1 and 4 and the complement thereof, wherein the 3′ end of said contiguous span is located at the 3′ end of said polynucleotide, and wherein the 3′ end of said polynucleotide is located within 20 nucleotides upstream of a biallelic marker of RBP-7 in said sequence; e) a polynucleotide consisting essentially of a sequence selected from the sequences of SEQ D) Nos. 72-101; f) a polynucleotide which encodes a polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ID No. 29; and g) a polynucleotide for use in hybridization assays, sequencing assays, and enzyme-based mismatch detection assays for determining the identity of the nucleotide at a biallelic marker of RBP-7.
 23. A recombinant vector comprising a polynucleotide selected from the group consisting of: a) a polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 1 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 1: 1-481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842-146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, and 161453-162450; b) a polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 4 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579; c) a polynucleotide having a sequence selected from the group of SEQ ID Nos. 5-28, a sequence complementary thereto or a fragment or a variant thereof; d) a polynucleotide comprising a combination of at least two polynucleotides selected from the group consisting of SEQ ID Nos. 5-28, wherein the polynucleotides are arranged within the nucleic acid, from the 5′ end to the 3′ end of said nucleic acid, in the same order than in SEQ ID No. 1; and e) a polynucleotide which encodes a polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ID No.
 29. 24. A host cell comprising a recombinant vector according to claim
 23. 25. A non-human host animal or mammal comprising a recombinant vector according to claim
 23. 26. A mammalian host cell comprising a RBP-7 gene disrupted by homologous recombination with a knock out vector, comprising a polynucleotide selected from the group consisting of: a) a polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 1 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 1:1481, 666-1465, 1521 67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406 87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842-146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, and 161453-162450; b) a polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 4 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350, 1703 1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579; c) a polynucleotide having a sequence selected from the group of SEQ ID Nos. 5-28, a sequence complementary thereto or a fragment or a variant thereof, d) a polynucleotide comprising a combination of at least two polynucleotides selected from the group consisting of SEQ ID Nos. 5-28, wherein the polynucleotides are arranged within the nucleic acid, from the 5′ end to the 3′ end of said nucleic acid, in the same order than in SEQ ID No. 1; and e) a polynucleotide which encodes a polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ID No.
 29. 27. A non-human host mammal comprising a RBP-7 gene disrupted by homologous recombination with a knock out vector, comprising a polynucleotide selected from the group consisting of: a) a polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 1 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 1: 1481, 666-1465, 1521-67592, 67704-71118, 71185-72598, 72690-75543, 75624-81841, 81934-83019, 83406-87901, 88041-93856, 93937-97158, 97236-98962, 99086-103188, 103745-104303, 104654-105084, 105180-106682, 106781-107798, 107897-108392, 108552-114335, 114418-114491, 114594-132246, 132332-134150, 134350-145565, 145842-146332, 146775-150446, 150542-152959, 153176-155590, 155738-159701, 160466-161028, and 161453-162450; b) a polynucleotide comprising a contiguous span of at least 12 nucleotides of SEQ ID No. 4 or the complements thereof, wherein said contiguous span comprises at least 1 of the following nucleotide positions of SEQ ID No. 4: 1-208, 1307-1350, 1703-1865, 2107-2180, 2843-3333, 3871-3882, 4222-4276, and 5017-5579; c) a polynucleotide having a sequence selected from the group of SEQ ID Nos. 5-28, a sequence complementary thereto or a fragment or a variant thereof; d) a polynucleotide comprising a combination of at least two polynucleotides selected from the group consisting of SEQ ID Nos. 5-28, wherein the polynucleotides are arranged within the nucleic acid, from the 5′ end to the 3′ end of said nucleic acid, in the same order than in SEQ ID No. 1; and e) a polynucleotide which encodes a polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ID No.
 29. 28. An isolated, purified, or recombinant polypeptide comprising a contiguous span of at least 6 amino acids of SEQ ID No.
 29. 29. An isolated or purified antibody composition capable of selectively binding to an epitope-containing fragment of a polypeptide according to claim
 28. 